EP3896452A1 - Method for assisting prediction of exacerbation of respiratory infection and reagent kit - Google Patents

Method for assisting prediction of exacerbation of respiratory infection and reagent kit Download PDF

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Publication number
EP3896452A1
EP3896452A1 EP21168754.6A EP21168754A EP3896452A1 EP 3896452 A1 EP3896452 A1 EP 3896452A1 EP 21168754 A EP21168754 A EP 21168754A EP 3896452 A1 EP3896452 A1 EP 3896452A1
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EP
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Prior art keywords
biomarker
respiratory infection
exacerbation
subject
ccl17
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EP21168754.6A
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German (de)
French (fr)
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EP3896452B1 (en
Inventor
Masashi Mizokami
Masaya SUGIYAMA
Norio OHMAGARI
Noriko KINOSHITA
Youichi Takahama
Kazuki Nakabayashi
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Sysmex Corp
National Center for Global Health and Medicine
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Sysmex Corp
National Center for Global Health and Medicine
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Priority claimed from JP2020101833A external-priority patent/JP6960642B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/125—Adult respiratory distress syndrome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a method for assisting exacerbation of respiratory infection.
  • the present invention relates to a reagent kit used for these methods.
  • An object of the present invention is to provide a new means for predicting exacerbation of respiratory infection by using such biomarker.
  • Japanese Examined Patent No. 6081699 describes that IL (Interleukin)-28B (also called IPN ⁇ 3), a type of cytokine, in serum of patient with chronic hepatitis C was detected by a method that specifically measures IL-28B.
  • IPN ⁇ 3 Interleukin-28B
  • Japanese Examined Patent No. 6081699 does not describe that IPN ⁇ 3 was measured in order to predict exacerbation of respiratory infection.
  • IFN ⁇ 3 Interferon ⁇ 3
  • CCL17 CC chemokine ligand 17
  • CXCL11 CXC chemokine ligand 11
  • IP-10 Interferon-inducible Protein-10
  • IL-6 Interferon-inducible Protein-10
  • CXCL9 CXC chemokine ligand 9
  • the present invention provides a method for assisting prediction of exacerbation of respiratory infection, comprising: measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, wherein the biomarker is at least one selected from the group consisting of IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and a measured value of the biomarker is an index to predict exacerbation of the respiratory infection.
  • the biomarker is IFN ⁇ 3, CXCL11, IP-10, IL-6 or CXCL9, and when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker is CCL17, and when the measured value of CCL17 is less than a threshold value corresponding to CCL17, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the index is a temporal change in the measured value of the biomarker in the subject.
  • the specimen is whole blood, plasma or serum.
  • the respiratory infection is an infection caused by a virus.
  • the virus is coronavirus or influenzavirus.
  • the virus is ⁇ -coronavirus, ⁇ -coronavirus, ⁇ -coronavirus or ⁇ -coronavirus.
  • the ⁇ -coronavirus is any one of SARS-CoV-2, HCoV-OC43, HCoV-HKU1, SARS-CoV, Bat SL-CoV-WIV1, BtCoV-HKU4, BtCoV-HKU5, MERS-CoV and BtCoV-HKU9.
  • the present invention provides a method for monitoring a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, comprising: acquiring measured values of the biomarker using specimens collected from the subject at a plurality of time points, wherein the biomarker is at least one selected from the group consisting of IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and the measured values are indices to predict exacerbation of the respiratory infection.
  • the biomarker is IFN ⁇ 3, CXCL11, IP-10, IL-6 or CXCL9, and when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker is CCL17, and when the measured value of CCL17 is less than a threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the present invention provides a use of a reagent kit for the method according to any one of claims 1 to 15, the reagent kit comprising a reagent comprising a substance capable of specifically binding to the biomarker.
  • Another aspect of the present invention is a method for assisting prediction of exacerbation of respiratory infection, comprising:
  • Another aspect of the present invention is a device to assist in predicting exacerbation of respiratory infection, comprising a computer comprising a processor and a memory under control of the processor, in which a computer program for making the computer executes calculating a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, and the output of the measured value of the biomarker is recorded in the memory, wherein the measured value of the biomarker is an index to predict exacerbation of the respiratory infection, and the biomarker comprises at least one selected from the group consisting of IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • Another aspect of the present invention is a computer program to assist in predicting exacerbation of respiratory infection.
  • This computer program is recorded on a computer-readable medium.
  • This computer program makes a computer execute calculating a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, and outputting the measured value of the biomarker.
  • the biomarker comprises at least one selected from the group consisting of IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • the method for assisting prediction of exacerbation of respiratory infection in a subject of the present invention (hereinafter, also referred to as "prediction method"), first, as a biomarker in a specimen collected from said subject, at least one selected from the group consisting of IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9 is measured.
  • the measured value of the biomarker serves as an index to predict exacerbation of the respiratory infection in said subject.
  • Respiratory infection refers to a disease caused by infection of respiratory organ such as nasal cavity, pharynx, trachea, bronchi or alveoli with a pathogen.
  • the pathogen is not particularly limited, and examples thereof include viruses, bacteria, fungi, parasites and the like.
  • examples of the virus include coronavirus, influenza virus, and the like.
  • the coronavirus is not particularly limited, and examples thereof include ⁇ -coronavirus, ⁇ -coronavirus, ⁇ -coronavirus, and ⁇ -coronavirus.
  • ⁇ -coronavirus examples include SARS-CoV-2, SARS-CoV, HCoV-OC43, HCoV-HKU1, Bat SL-CoV-WIV1, BtCoV-HKU4, BtCoV-HKU5, MERS-CoV, BtCoV-HKU9, and the like.
  • Examples of the subject in the methods of the present invention include a patient suffering from a respiratory infection and a person suspected of having a respiratory infection.
  • patient suffering from a respiratory infection refers to a person whose respiratory infection has been confirmed due to detection of a pathogen or the like and has not yet become severe.
  • the person suspected of having a respiratory infection include, for example, those who have cold symptoms such as fever, cough, runny nose and sore throat and/or symptoms specific to prescribed respiratory infections such as feeling of dyspnea, short breath during exertion and abnormal taste and smell, and those having come into contact with and those suspected of having come into contact with patient with respiratory infection.
  • Contact with patient with respiratory infection refers to, for example, an act such as talking with the patient within a distance of 1 m, staying in a closed space where the patient is present, and being contacted with saliva, coughing or the like of the patient.
  • the reference to "exacerbation of respiratory infection” refers to developing pneumonia requiring oxygen inhalation or being in a condition requiring intensive care management including mechanical ventilation management.
  • the specimen used in the methods of the invention is not particularly limited as long as it is a liquid sample collected from a subject and suspected of containing the biomarker.
  • liquid sample include blood samples, cerebrospinal fluid, sputum, bronchoalveolar lavage fluid, nasopharyngeal swab, lymph fluid, urine, stool, saliva, and the like.
  • blood samples are preferred.
  • the blood sample include whole blood, plasma and serum, and plasma and serum are particularly preferable.
  • impurities may be removed from the specimen by a known means such as centrifugal separation and filtration.
  • the specimen may be diluted with an appropriate aqueous medium as necessary.
  • an aqueous medium is not particularly limited as long as it does not interfere with the measurement described herein below, and examples thereof include water, physiological saline, a buffer solution, and the like.
  • the buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutrality (for example, a pH of 6 or more and 8 or less). Examples of the buffer solution include Good buffers such as HEPES, MES, Tris and PIPES, phosphate buffered saline (PBS), and the like.
  • the biomarker measured by the prediction method of the present invention is one or more protein molecules selected from IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • IFN ⁇ 3, also called IL-28B is a protein consisting of 200 amino acids encoded by a gene of about 1.5 Kb located on chromosome 19.
  • IFN ⁇ 3 has 25 signal peptides at N-terminus, and the signal peptide is cleaved when IPN ⁇ 3 is secreted extracellularly.
  • CCL17 is also called TARC (Thymus- and activation-regulated chemokine) and is a type of Th2 type chemokine.
  • CXCL11 is a chemokine also called I-TAC (Interferon-inducible T-cell Chemoattractant), and is a ligand for CXCR3 receptor.
  • IP-10 also called CXCL10, is a type of Thl type chemokine.
  • IL-6 is a type of TH2 type cytokine.
  • CXCL9 also called MIG (Monokine induced by interferon ⁇ ), is a ligand for CXCR3 receptor and is a type of Thl type chemokine as well as IP-10.
  • MIG Monokine induced by interferon ⁇
  • IFN ⁇ 3 may have an amino acid sequence represented by SEQ ID NO: 1 or may have an amino acid sequence represented by SEQ ID NO: 2.
  • biomarkers selected from IFN ⁇ 3, CCL17, CXCL11, IP-10, IL-6 and CXCL9 from the viewpoint of improving accuracy of prediction of exacerbation.
  • biomarkers include one of the following combinations:
  • any known method can be used for measuring the biomarker, and the method is not particularly limited.
  • a method of capturing a biomarker using a substance capable of specifically binding to the biomarker is preferable.
  • the biomarker contained in the specimen can be measured by detecting the biomarker captured by such a substance by a method known in the art.
  • the measured value of the biomarker may be a value that reflects an amount or concentration in the specimen.
  • the measured value may be a concentration or a value reflecting the concentration calculated based on a measurement result of a calibrator.
  • the "value reflecting the concentration" depends on a type of labeling substance described herein below, and examples thereof include a measured value of fluorescence intensity, a measured value of emission intensity, a measured value of radioactivity, and the like.
  • Examples of the substance capable of specifically binding to the biomarker include an antibody, an aptamer and the like, among which an antibody is particularly preferable.
  • An antibody against the biomarker is not particularly limited as long as it is an antibody capable of specifically binding to the biomarker.
  • Such an antibody may be any of monoclonal antibodies, polyclonal antibodies, and fragments thereof (for example, Fab, F(ab')2, etc.). A commercially available antibody may be used.
  • a monoclonal antibody having a heavy chain variable region domain and a light chain variable region domain described in any one of (1) to (5) below or a fragment thereof may be used.
  • the method used for measuring a biomarker using an antibody is not particularly limited and can be appropriately selected from known immunoassays.
  • an enzyme-linked immunosorbent assay (ELISA method) is preferred, and a sandwich ELISA method being particularly preferred.
  • ELISA method enzyme-linked immunosorbent assay
  • sandwich ELISA method being particularly preferred.
  • a complex containing a biomarker, an antibody for capturing the biomarker (hereinafter also referred to as “capture antibody”) and an antibody for detecting the biomarker (hereinafter also referred to as “detection antibody”) is formed and/or captured on a solid phase.
  • a complex can be formed by mixing the specimen, a capture antibody, and a detection antibody. Then, a solution containing the complex is brought into contact with a solid phase capable of immobilizing the capture antibody, whereby the complex can be captured on the solid phase.
  • a solid phase preliminarily immobilized with the capture antibody may be used.
  • a solid phase immobilized with the capture antibody, the specimen, and the detection antibody are brought into contact with each other, whereby the complex can be formed on the solid phase.
  • both the capture antibody and the detection antibody are monoclonal antibodies, it is preferable that the epitopes be different from each other.
  • the mode of immobilization of the capture antibody on the solid phase is not particularly limited.
  • the capture antibody and the solid phase may be bound directly, or the capture antibody and the solid phase may be indirectly bound via another substance.
  • Examples of the direct binding include physical adsorption and the like.
  • Examples of the indirect bond include a bond via a combination of biotins and avidins.
  • the biotins include biotin and biotin analogs such as desthiobiotin.
  • the avidins include avidin and analogs of avidins such as streptavidin and tamavidin (registered trademark).
  • the material of the solid phase is not particularly limited.
  • the material can be selected from organic polymer compounds, inorganic compounds, biopolymers, and the like.
  • the organic polymer compound include latex, polystyrene, polypropylene, and the like.
  • the inorganic compound include magnetic bodies (iron oxide, chromium oxide, ferrite, and the like), silica, alumina, glass, and the like.
  • the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose, and the like. Two or more of these may be used in combination.
  • the shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, test tubes, and the like. Among them, particles are preferable, and magnetic particles are particularly preferable.
  • B/F (Bound/Free) separation for removing an unreacted free component not forming a complex may be performed between the process of forming the complex and the process of detecting the complex.
  • the unreacted free component refers to a component not constituting a complex. Examples thereof include capture antibodies not bound to the biomarker, detection antibodies, and the like.
  • the means of B/F separation is not particularly limited, and when the solid phase is a particle, B/F separation can be performed by recovering only the solid phase capturing the complex by centrifugation. When the solid phase is a container such as a microplate or a microtube, B/F separation can be performed by removing a liquid containing an unreacted free component.
  • B/F separation can be performed by aspirating and removing a liquid containing an unreacted free component by a nozzle while magnetically constraining the magnetic particles with a magnet, which is preferable from the viewpoint of automation.
  • the solid phase capturing the complex may be washed with a suitable aqueous medium such as PBS.
  • a measured value of a biomarker contained in the specimen can be acquired by detecting the complex formed on the solid phase by a method known in the art.
  • the measured value of the marker in the liquid sample can be acquired by detecting a signal generated by the labeling substance.
  • the measured value of the biomarker in the liquid sample can be acquired in the same manner.
  • the immune complex transfer method described in Japanese Laid-Open Patent Publication No. H01-254868 can be also used.
  • detecting a signal herein includes qualitatively detecting the presence or absence of a signal, quantifying a signal intensity, and semi-quantitatively detecting the intensity of a signal.
  • the labeling substance is not particularly limited.
  • the labeling substance may be a substance which itself generates a signal (hereinafter also referred to as "signal generating substance") or a substance which catalyzes the reaction of other substances to generate a signal.
  • the signal generating substance include fluorescent substances, radioactive isotopes, and the like.
  • the substance that catalyzes the reaction of other substances to generate a detectable signal include enzymes.
  • the enzymes include alkaline phosphatase, peroxidase, ⁇ -galactosidase, luciferase, and the like.
  • fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), fluorescent proteins such as GFP, and the like.
  • fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), fluorescent proteins such as GFP, and the like.
  • radioactive isotopes include 125 I, 14 C, 32 P, and the like.
  • an enzyme is preferable as a labeling substance, and alkaline phosphatase and peroxidase are particularly preferable.
  • a measurement method according to the type of signal derived from the labeling substance may be appropriately selected.
  • the labeling substance is an enzyme
  • signals such as light and color generated by reacting a substrate for the enzyme can be measured by using a known apparatus such as a spectrophotometer.
  • the substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme.
  • examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (disodium 4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate) and CSPD (registered trademark) (disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indo
  • BCIP 5-bromo-4-chloro-3-indoly
  • examples of the substrate include chemiluminescent substrates such as luminol and derivatives thereof, and chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB).
  • chemiluminescent substrates such as luminol and derivatives thereof
  • chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB).
  • the labeling substance is a radioactive isotope
  • radiation as a signal can be measured using a known apparatus such as a scintillation counter.
  • fluorescence as a signal can be measured using a known apparatus such as a fluorescence microplate reader.
  • the excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.
  • the detection result of the signal can be used as the measured value of the biomarker.
  • the signal intensity value itself or a value acquired from the measured value can be used as the measured value of the biomarker.
  • the value acquired from the measured value of the signal intensity include a value acquired by subtracting the measured value of a negative control sample or the background value from the measured value, a value acquired by applying the measured value to a calibration curve, and the like.
  • the negative control sample can be appropriately selected, and examples thereof include a specimen obtained from a mildly ill patient (for example, an infectious disease patient who has recovered without becoming severe), a specimen obtained from a healthy person, and the like.
  • biomarker contained in the specimen by an immunoassay such as an EIA method or an ELISA method.
  • Biomarkers can be measured using commercially available devices and reagents such as the HISCL series (manufactured by Sysmex Corporation) and the Bio-Plex Multiplex System (manufactured by Bio-Rad Laboratories, Inc.).
  • the measured values of the biomarkers described above can be used as an index indicating whether or not a respiratory infection of a subject is exacerbated or likely to exacerbate. For example, by comparing the acquired measured value of the biomarker with a threshold value corresponding to the biomarker (i.e. a threshold value previously established for the biomarker), the measured value of the biomarker may be used as an index suggesting that the possibility or likeliness of exacerbation of respiratory infection of the subject is high or low.
  • a threshold value corresponding to the biomarker i.e. a threshold value previously established for the biomarker
  • the possibility or likeliness of exacerbation of the respiratory infection is a risk of exacerbation of the respiratory infection of the subject after a lapse of a predetermined period or within a predetermined period (for example, 1 day to 1 month) from a date when the specimen was collected from the subject.
  • IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9 showed high values in a patient with severe respiratory infection and low values in a patient with mild respiratory infection.
  • Measured values of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9 can be used as indexes suggesting the possibility or likeliness of exacerbation of respiratory infection, by comparison with threshold values corresponding to each biomarker (i.e. for each biomarker).
  • threshold values corresponding to each biomarker i.e. for each biomarker.
  • the biomarker contains IFN ⁇ 3 and the measured value of IFN ⁇ 3 is greater than or equal to a threshold value corresponding to IFN ⁇ 3, the possibility or likeliness of exacerbation of respiratory infection in the subject is suggested to be high.
  • the measured value of IFN ⁇ 3 is less than the threshold value corresponding to IFN ⁇ 3, the possibility or likeliness of exacerbation of respiratory infection in the subject is suggested to be low.
  • the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to a threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of CXCL11 is less than the threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to a threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of IP-10 is less than the threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to a threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of IL-6 is less than the threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to a threshold value corresponding to CXCL9
  • the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high
  • the measured value of CXCL9 is less than the threshold value corresponding to CXCL9
  • the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains at least two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be suggested by their measured values. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When all of the measured values of the biomarkers selected from the group are less than threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarkers are two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and either or both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are three selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are four selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are five consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6, and CXCL9 and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarker contains at least two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject can be classified into three stages by their measured values. Specifically:
  • the biomarkers are two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are three selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are four selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the biomarkers are five consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • CCL17 in the specimen may be measured.
  • CCL17 unlike other biomarkers, showed a low value in a patient with severe respiratory infection and a high value in a patient with mild respiratory infection.
  • the measured value of CCL17 can be used as an index suggesting the possibility of exacerbation of respiratory infection, by comparison with a threshold value corresponding to CCL17.
  • the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • CCL17 is used in combination with at least one biomarker selected from IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9.
  • the biomarker contains at least one selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be suggested by their measured values.
  • the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains at least one selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject can be classified into three stages by their measured values. Specifically:
  • biomarkers contain IFN ⁇ 3 and CCL17,
  • the threshold values corresponding to each biomarker are not particularly limited and can be set as appropriate.
  • specimens are collected from a plurality of patients with respiratory infection, and biomarkers in the specimens are measured to obtain measured values. After a predetermined period (for example, 2 weeks) has passed since the specimens were collected, whether or not the respiratory infection has become severe in these subjects is confirmed.
  • the acquired measured value data is classified into data of a group of severely ill patients and data of a group of non-severely ill patients. Then, for each biomarker, a value that can most accurately distinguish between the group of severely ill patients and the group of non-severely ill patients is determined, and the value is set as a threshold value.
  • the threshold value it is possible to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.
  • the threshold value corresponding to IFN ⁇ 3 (or for IFN ⁇ 3) is set in the range of, for example, 4 pg/mL or more and 15 pg/mL or less, i.e. the threshold is a value between 4 and 15 pg/mL.
  • the threshold value corresponding to CXCL11 is set in the range of, for example, 20 pg/mL or more and 40 pg/mL or less, i.e. the threshold is a value between 20 and 40 pg/mL.
  • the threshold value corresponding to IP-10 is set in the range of, for example, 400 pg/mL or more and 1200 pg/mL or less, i.e.
  • the threshold is a value between 400 and 1200 pg/mL.
  • the threshold value corresponding to IL-6 is set in the range of, for example, 4 pg/mL or more and 6 pg/mL or less, i.e. the threshold is a value between 4 and 6 mL.
  • the threshold value corresponding to CXCL9 is set in the range of, for example, 30 pg/mL or more and 40 pg/mL or less, i.e. the threshold is a value between 30 and 40 pg/mL.
  • the threshold value corresponding to CCL17 is set in the range of, for example, 40 pg/mL or more and 100 pg/mL or less, i.e. the threshold is a value between 40 and 100 pg/mL.
  • the "other information” includes findings on X-ray or CT images of the lungs and other medical findings.
  • the biomarker when used as an index, this may refer to the absolute value of the biomarker in the sample of the patient. Additionally or alternatively, as an index to predict exacerbation of the respiratory infection of the subject in particular embodiments, temporal change of the measured value of the biomarker in the subject may be acquired.
  • the temporal change of the measured value of the biomarker is not particularly limited as long as it is information showing transition of the measured value of the biomarker in the specimen collected from the subject a plurality of times periodically or irregularly over time. Examples of such temporal change include values calculated from a plurality of measured values (for example, the difference, ratio, etc.
  • the methods of the present invention involve determining the biomarker in two or more specimens collected from the subject at different time points and determining the temporal change of the value of said biomarker.
  • the methods of the present invention involve determining the biomarker in two or more specimens collected from the subject at different time points and determining the likeliness of exacerbation based on said two or more measurements, as detailed for the method of monitoring described below.
  • the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high based on the measured value of the biomarker, it is possible to perform medical intervention to prevent the exacerbation of respiratory infection on the subject.
  • the medical intervention include drug administration, surgery, immunotherapy, gene therapy, oxygenation procedures, heart-lung machine procedures, and the like.
  • the drug can be appropriately selected from known therapeutic drugs for respiratory infections or candidate medicines therefor.
  • the respiratory infection is SARS-CoV-2 infection
  • examples of the drug include drugs having an antiviral action, drugs that reduce inflammation, ACE inhibitors, and the like.
  • the drug examples include favipiravir, lopinavir, ritonavir, nafamostat, camostat, remdesivir, ribavirin, ivermectin, ciclesonide, chloroquine, hydroxychloroquine, interferon, tocilizumab, sarilumab, tofasitinib, baricitinib, ruxolitinib, acalabrutinib, ravulizumab, eritoran, ibudilast, HLCM051, LY3127804, and the like.
  • the prediction method of the present invention may include predicting exacerbation of respiratory infection, based on a measured value of a biomarker determined in the specimen collected from the subject.
  • the measured value of the biomarker is compared with a threshold value corresponding to the biomarker, and based on the comparison result, it may be determined whether the possibility or likeliness of exacerbation of respiratory infection of the subject is high or low. Details of the threshold value corresponding to the biomarker are as described above.
  • the biomarker contains IFN ⁇ 3 and the measured value of IFN ⁇ 3 is greater than or equal to the threshold value corresponding to IFN ⁇ 3, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of IFN ⁇ 3 is less than the threshold value corresponding to IFN ⁇ 3, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to the threshold value corresponding to CXCL11, the possibility of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of CXCL11 is less than the threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to the threshold value corresponding to IP-10, the possibility of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of IP-10 is less than the threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to the threshold value corresponding to IL-6, the possibility of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of IL-6 is less than the threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to the threshold value corresponding to CXCL9, the possibility of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of CXCL9 is less than the threshold value corresponding to CXCL9, the possibility of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains at least two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be determined based on their measured values. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. When all of the measured values of the biomarkers selected from the group are less than the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarkers are two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and either or both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the biomarkers are three selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the biomarkers when the biomarkers are four selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. In another example, when the biomarkers are five consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the biomarker contains at least two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be determined in three stages based on their measured values. Specifically:
  • the biomarkers are two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high.
  • the biomarkers are three selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high.
  • the biomarkers are four selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high.
  • the biomarkers are five consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high.
  • the biomarker contains at least one selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be determined based on their measured values in a sample of said subject. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, and/or the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • the biomarker contains at least one selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be determined in three stages based on their measured values. Specifically:
  • biomarkers contain IFN ⁇ 3 and CCL17,
  • the application thus also provides methods for monitoring a measured value of a biomarker in a specimen collected from a subject (hereinafter, also referred to as "monitoring method"). These methods are of particular interest in subjects suffering from a respiratory infection and a person suspected of having a respiratory infection and can be used to monitor the risk of exacerbation of respiratory infection of the subject over time.
  • the measured value of the biomarker is acquired using specimens collected from the subject at a plurality of time points. Details of the subject, the specimen, the biomarker and acquisition of the measured value thereof are the same as those described for the prediction method of the present embodiment described above.
  • the plurality of time points may be two or more different time points.
  • the plurality of time points includes a first time point and a second time point different from the first time point.
  • the first time point is not particularly limited and can be any time point.
  • the first time point may be a time point when the subject is found to have a respiratory infection, a time point when the subject develops symptoms of a respiratory infection, a time point when the subject is hospitalized, or the like
  • the second time point is not particularly limited as long as it differs from the first time point.
  • the second time point is a time point when a period within one month has passed from the first time point.
  • the second time point is a time point when 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 2 weeks, 3 weeks, 4 weeks or one month has passed from the first time point.
  • the "specimens collected from the subject at a plurality of time points" are specimens collected from the same subject at each of the plurality of time points.
  • it includes a first specimen collected from a subject at a first time point and a second specimen collected from the subject at a second time point different from the first time point.
  • the biomarker may be measured each time a specimen is collected, or each collected specimen may be stored and measured collectively.
  • the measured value of the biomarker in the same subject is monitored and used as an index to predict exacerbation of the respiratory infection.
  • the measured values of the same biomarker at a plurality of time points are acquired.
  • the measured value of the biomarker measured from each specimen is compared with the threshold value corresponding to the biomarker, and based on the comparison result, the possibility, also referred to as the the risk or likeliness, of exacerbation of respiratory infection of the subject may be suggested to be high or low. Details of the threshold value corresponding to the biomarker are similar to those described for the prediction methods described above.
  • the biomarker contains IFN ⁇ 3 and the measured value of IFN ⁇ 3 is greater than or equal to the threshold value corresponding to IFN ⁇ 3 at at least one of the plurality of time points, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of IFN ⁇ 3 is less than the threshold value corresponding to IFN ⁇ 3 at any of the plurality of time points, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to the threshold value corresponding to CXCL11 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of CXCL11 is less than the threshold value corresponding to CXCL11 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to the threshold value corresponding to IP-10 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of IP-10 is less than the threshold value corresponding to IP-10 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to the threshold value corresponding to IL-6 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of IL-6 is less than the threshold value corresponding to IL-6 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to the threshold value corresponding to CXCL9 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of CXCL9 is less than the threshold value corresponding to CXCL9 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains at least two selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be suggested by the measured values of the biomarkers at a plurality of time points.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the measured value of CCL17 in each of the specimens taken from the subject may be acquired.
  • the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • the biomarker contains at least one selected from the group consisting of IFN ⁇ 3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be suggested by the measured values of the biomarkers at a plurality of time points. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, and/or the measured value of CCL17 is less than the threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • the conditions for terminating the monitoring method of the present invention are not particularly limited, and a healthcare professional such as a doctor may appropriately determine the conditions.
  • a healthcare professional such as a doctor may appropriately determine the conditions.
  • the monitoring method of the present embodiment may be terminated.
  • the monitoring method of the present embodiment may be terminated. Additionally, or alternatively the methods of the present invention such as the diagnosis or monitoring methods described herein may be used to determine which treatment will be applied.
  • the invention also provides a reagent kit for use in the prediction methods and/or monitoring methods of the present invention described above.
  • the reagent kit of the present invention includes at least one reagent selected from a reagent containing a substance capable of specifically binding to IFN ⁇ 3, a reagent containing a substance capable of specifically binding to CCL17, a reagent containing a substance capable of specifically binding to CXCL11, a reagent containing a substance capable of specifically binding to IP-10, a reagent containing a substance capable of specifically binding to IL-6, and a reagent containing a substance capable of specifically binding to CXCL9.
  • the reagent kit may include at least one reagent selected from the reagent containing a substance capable of specifically binding to IFN ⁇ 3, the reagent containing a substance capable of specifically binding to CXCL11, the reagent containing a substance capable of specifically binding to IP-10, the reagent containing a substance capable of specifically binding to IL-6, and the reagent containing a substance capable of specifically binding to CXCL9, and the reagent containing a substance capable of specifically binding to CCL17.
  • the reagent kit includes the reagent containing a substance capable of specifically binding to IFN ⁇ 3 and the reagent containing a substance capable of specifically binding to CCL17.
  • the substance capable of specifically binding to each biomarker include an antibody, an aptamer, and the like. The antibody is particularly preferable among them.
  • Fig. 1A shows an example of the reagent kit of the present embodiment.
  • 11 denotes a reagent kit
  • 12 denotes a first container containing a reagent containing a substance capable of specifically binding to IFN ⁇ 3
  • 13 denotes a second container containing a reagent containing a substance capable of specifically binding to CCL17
  • 14 denotes a packing box
  • 15 denotes an attached document. Composition, usage, storage method, etc. of each reagent may be described in the attached document.
  • the reagent kit of this example includes the reagent containing a substance capable of specifically binding to IFN ⁇ 3 and the reagent containing a substance capable of specifically binding to CCL17, but in place of these reagents, reagents containing a substance capable of specifically binding to another biomarker may be included.
  • the reagent kit of the present embodiment includes a capture antibody and a detection antibody for the biomarker.
  • the detection antibody may be labeled with a labeling substance. Details of the capture antibody, the detection antibody and the labeling substance are the same as those described for the prediction method of the present embodiment.
  • the reagent kit may include a solid phase and a substrate. Details of the solid phase and the substrate are the same as those described for the prediction method of the present embodiment.
  • Fig. 1B shows an example of a reagent kit of a further embodiment.
  • 21 denotes a reagent kit
  • 22 denotes a first container containing a reagent containing a capture antibody for IFN ⁇ 3
  • 23 denotes a second container containing a reagent containing a detection labeled antibody for IFN ⁇ 3
  • 24 denotes a third container containing a reagent containing a capture antibody for CCL17
  • 25 denotes a fourth container containing a reagent containing a detection labeled antibody for CCL17
  • 26 denotes an attached document
  • 27 denotes a packing box.
  • the reagent kit of this example includes reagents each containing the capture antibody and the detection labeled antibody for IFN ⁇ 3 and reagents each containing the capture antibody and the detection labeled antibody for CCL17, but in place of these reagents, reagents each containing a capture antibody for another biomarker and a detection labeled antibody for another biomarker may be included.
  • any of the above reagent kits include a calibrator.
  • the calibrator include a calibrator for quantifying IFN ⁇ 3 (calibrator for IFN ⁇ 3), a calibrator for quantifying CXCL11 (calibrator for CXCL11), a calibrator for quantifying IP-10 (calibrator for IP-10), a calibrator for quantifying IL-6 (calibrator for IL-6), a calibrator for quantifying CXCL9 (calibrator for CXCL9), and a calibrator for quantifying CCL17 (calibrator for CCL17).
  • the calibrator for IFN ⁇ 3 may include, for example, a buffer solution containing no IFN ⁇ 3 (negative control) and a buffer solution containing IFN ⁇ 3 at a known concentration.
  • the calibrator for CXCL11 may include, for example, a buffer solution containing no CXCL11 (negative control) and a buffer solution containing CXCL11 at a known concentration.
  • the calibrator for IP-10 may include, for example, a buffer solution containing no IP-10 (negative control) and a buffer solution containing IP-10 at a known concentration.
  • the calibrator for IL-6 may include, for example, a buffer solution containing no IL-6 (negative control) and a buffer solution containing IL-6 at a known concentration.
  • the calibrator for CXCL9 may include, for example, a buffer solution containing no CXCL9 (negative control) and a buffer solution containing CXCL9 at a known concentration.
  • the calibrator for CCL17 may include, for example, a buffer solution containing no CCL17 (negative control) and a buffer solution containing CCL17 at a known concentration.
  • Fig. 1C shows an example of a reagent kit of a further embodiment.
  • 31 denotes a reagent kit
  • 32 denotes a first container containing a reagent containing a capture antibody for IFN ⁇ 3
  • 33 denotes a second container containing a reagent containing a detection labeled antibody for IFN ⁇ 3
  • 34 denotes a third container containing a reagent containing a capture antibody for CCL17
  • 35 denotes a fourth container containing a reagent containing a detection labeled antibody for CCL17
  • 36 denotes a fifth container containing a buffer solution containing neither IFN ⁇ 3 nor CCL17
  • 37 denotes a sixth container containing a buffer solution containing IFN ⁇ 3 and CCL17 at each predetermined concentrations
  • 38 denotes a packing box
  • 39 denotes an attached document.
  • a buffer solution containing neither IPN ⁇ 3 nor CCL17 and a buffer solution containing IFN ⁇ 3 and CCL17 at each predetermined concentration can be used as a calibrator for quantifying IFN ⁇ 3 and CCL17.
  • the reagent kit of this example includes reagents each containing the capture antibody and the detection labeled antibody for IFN ⁇ 3, reagents each containing the capture antibody and the detection labeled antibody for CCL17 and a calibrator for quantifying IFN ⁇ 3 and CCL17, but in place of these reagents, reagents each containing a capture antibody for another biomarker and a detection labeled antibody for another biomarker and a calibrator may be included.
  • a container containing one reagent selected from the reagent containing a substance capable of specifically binding to IFN ⁇ 3, the reagent containing a substance capable of specifically binding to CCL17, the reagent containing a substance capable of specifically binding to CXCL11, the reagent containing a substance capable of specifically binding to IP-10, the reagent containing a substance capable of specifically binding to IL-6, and the reagent containing a substance capable of specifically binding to CXCL9 may be packed in a box and provided to a user as a reagent kit.
  • the box may contain an attached document. Compositions, usage, storage method, etc. of the reagents may be described in the attached document. Fig.
  • FIG.1D shows an example of the reagent kit.
  • 41 denotes a reagent kit
  • 42 denotes a container containing a reagent containing a substance capable of specifically binding to IFN ⁇ 3
  • 43 denotes a packing box
  • 44 denotes an attached document.
  • the reagent kit of this example includes the reagent containing a substance capable of specifically binding to IFN ⁇ 3, but in place of this reagent, reagents containing a substance capable of specifically binding to another biomarker may be included.
  • the application also provides a device for performing the prediction method and/or monitoring method of the present embodiment.
  • a device is a device to assist in predicting exacerbation of respiratory infection (hereinafter, also simply referred to as "device").
  • a computer program for making a computer execute the prediction method and/or monitoring method of the present invention.
  • Such a computer program is a computer program to assist in predicting exacerbation of respiratory infection.
  • Such a computer program when executed on a computer, causes the computer to carry out the methods of the invention.
  • FIG. 2 is a schematic diagram of a device of the present embodiment.
  • a device 10 shown in Fig. 2 includes an immunoassay device 20 and a computer system 30 connected to the immunoassay device 20.
  • the type of immunoassay device is not particularly limited, and it can be appropriately selected according to the method for measuring a biomarker.
  • the immunoassay device 20 is a commercially available automated immunoassay device capable of detecting a chemiluminescent signal generated by a sandwich ELISA method using magnetic particles on which a capture antibody is immobilized and an enzyme-labeled detection antibody.
  • the immunoassay device 20 is not particularly limited as long as it can detect a signal based on the used labeling substance, and it can be appropriately selected according to the type of the labeling substance.
  • the immunoassay device 20 When a reagent containing magnetic particles on which a capture antibody is immobilized, a reagent containing an enzyme-labeled detection antibody and a specimen collected from a subject are set in the immunoassay device 20, the immunoassay device 20 performs an antigen-antibody reaction using each reagent, acquires a chemiluminescent signal as optical information based on the enzyme-labeled antibody specifically bound to a biomarker, and transmits the obtained optical information to the computer system 30.
  • the computer system 30 includes a computer main body 300, an input unit 301, and a display unit 302 that displays specimen information, a determination result, and the like.
  • the computer system 30 receives the optical information from the immunoassay device 20.
  • a processor of the computer system 30 executes a computer program to assist in predicting exacerbation of respiratory infection, installed in a hard disk 313, based on the optical information.
  • the computer system 30 may be equipment separate from the immunoassay device 20, or may be equipment including the immunoassay device 20. In the latter case, the computer system 30 may itself be the prediction assisting device 10.
  • a commercially available automated immunoassay device may be loaded with the computer program to assist in predicting exacerbation of respiratory infection.
  • the device 10 may be a device in which the immunoassay device 20 and the computer system 30 are integrally configured.
  • the computer main body 300 includes a central processing unit (CPU) 310, a read only memory (ROM) 311, a random access memory (RAM) 312, a hard disk 313, an input/output interface 314, a reading device 315, a communication interface 316, and an image output interface 317.
  • the CPU 310, the ROM 311, the RAM 312, the hard disk 313, the input/output interface 314, the reading device 315, the communication interface 316 and the image output interface 317 are data-communicably connected by a bus 318.
  • the immunoassay device 20 is communicably connected to the computer system 30 via the communication interface 316.
  • the CPU 310 can execute a program stored in the ROM 311 or the hard disk 313 and a program loaded in the RAM 312.
  • the CPU 310 calculates the measured value of the biomarker and displays it on the display unit 302.
  • the ROM 311 includes a mask ROM, PROM, EPROM, EEPROM, and the like.
  • ROM 311 a computer program executed by the CPU 310 and data used for executing the computer program are recorded as described above.
  • the computer program recorded in ROM 311 includes a basic input output system (BIOS).
  • the RAM 312 includes SRAM, DRAM, and the like.
  • the RAM 312 is used for reading the program recorded in the ROM 311 and the hard disk 313.
  • the RAM 312 is also used as a work area of the CPU 310 when these programs are executed.
  • an operating system and a computer program such as an application program to be executed by the CPU 310, and data used for executing the computer program are installed.
  • the reading device 315 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid state drive, and the like.
  • the reading device 315 can read a program or data recorded on a portable recording medium 40.
  • the input/output interface 314 includes, for example, a serial interface such as USB, IEEE1394 and RS-232C, a parallel interface such as SCSI, IDE and IEEE1284, and an analog interface including a D/A converter, an A/D converter and the like.
  • the input unit 301 such as a keyboard and a mouse is connected to the input/output interface 314. An operator can input various commands to the computer main body 300 through the input unit 301.
  • the communication interface 316 is, for example, an Ethernet (registered trademark) interface or the like.
  • the computer main body 300 can also transmit print data to a printer or the like through the communication interface 316.
  • the image output interface 317 is connected to the display unit 302 including an LCD, a CRT, and the like. As a result, the display unit 302 can output a video signal corresponding to the image data coming from the CPU 310.
  • the display unit 302 displays an image (screen) according to the input video signal.
  • a processing procedure to be executed by the device 10 of the present invention will be described with reference to Fig. 4A .
  • a measured value of IFN ⁇ 3 is acquired from a chemiluminescent signal generated by a sandwich ELISA method using magnetic particles on which a capture antibody is immobilized, and an enzyme-labeled detection antibody and output.
  • a measured value of CCL17, CXCL11, IP-10, IL-6 or CXCL9 may be acquired.
  • step S101 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates a measured value of IFN ⁇ 3 from the acquired optical information, and the CPU 310 stores the measured value of IFN ⁇ 3 in the hard disk 313.
  • step S103 the CPU 310 outputs the measured value of IFN ⁇ 3, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the measured value.
  • a threshold value corresponding to INF ⁇ 3 may also be displayed on the display unit 302 as reference information.
  • step S201 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates measured values of IFN ⁇ 3 and CCL17 from the acquired optical information, and the CPU 310 stores the measured values of IFN ⁇ 3 and CCL17 in the hard disk 313.
  • step S203 the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the measured values.
  • threshold values corresponding to each of IFN ⁇ 3 and CCL17 may also be displayed on the display unit 302 as reference information. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • step S301 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates a measured value of IFN ⁇ 3 from the acquired optical information, and the CPU 310 stores the measured value of IFN ⁇ 3 in the hard disk 313.
  • step S303 the CPU 310 compares the calculated measured value of IPN ⁇ 3 with a threshold value corresponding to IFN ⁇ 3 stored in the hard disk 313. When the measured value of IFN ⁇ 3 is greater than or equal to the threshold value, the process proceeds to step S304.
  • step S304 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S303 when the measured value of IFN ⁇ 3 is less than the threshold value, the process proceeds to step S305.
  • step S305 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • step S306 the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • Fig. 4D A flow for predicting exacerbation of respiratory infection based on the measured values of IFN ⁇ 3 and CXCL11 will be described with reference to Fig. 4D .
  • the threshold value corresponding to the measured value of IFN ⁇ 3 is referred to as "first threshold value”
  • the threshold value corresponding to the measured value of CXCL11 is referred to as "second threshold value”.
  • step S401 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates measured values of IFN ⁇ 3 and CXCL11 from the acquired optical information, and the CPU 310 stores the measured values of IFN ⁇ 3 and CXCL11 in the hard disk 313.
  • the CPU 310 compares the calculated measured value of IFN ⁇ 3 with the first threshold value stored in the hard disk 313. When the measured value of IFN ⁇ 3 is greater than or equal to the first threshold value, the process proceeds to step S404.
  • step S404 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S403 when the measured value of IFN ⁇ 3 is less than the first threshold value, the process proceeds to step S405.
  • the CPU 310 compares the calculated measured value of CXCL11 with the second threshold value stored in the hard disk 313. When the measured value of CXCL11 is greater than or equal to the second threshold value, the process proceeds to step S404, and the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S405 when the measured value of CXCL11 is less than the second threshold value, the process proceeds to step S406.
  • step S406 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • step S407 the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • Fig. 4E A flow for predicting exacerbation of respiratory infection based on the measured values of IFN ⁇ 3 and CCL17 will be described with reference to Fig. 4E .
  • the threshold value corresponding to the measured value of IFN ⁇ 3 is referred to as "first threshold value”
  • the threshold value corresponding to the measured value of CCL17 is referred to as "third threshold value”.
  • step S501 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates measured values of IFN ⁇ 3 and CCL17 from the acquired optical information, and the CPU 310 stores the measured values of IFN ⁇ 3 and CCL17 in the hard disk 313.
  • the CPU 310 compares the calculated measured value of IFN ⁇ 3 with the first threshold value stored in the hard disk 313. When the measured value of IFN ⁇ 3 is greater than or equal to the first threshold value, the process proceeds to step S504.
  • step S504 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S503 when the measured value of IFN ⁇ 3 is less than the first threshold value, the process proceeds to step S505.
  • the CPU 310 compares the calculated measured value of CCL17 with the third threshold value stored in the hard disk 313. When the measured value of CCL17 is greater than or equal to the third threshold value, the process proceeds to step S506.
  • step S506 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • step S505 when the measured value of CCL17 is less than the third threshold value, the process proceeds to step S504, and the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S507 the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • step S601 the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20.
  • the CPU 310 calculates a measured value of CCL17 from the acquired optical information, and the CPU 310 stores the measured value of CCL17 in the hard disk 313.
  • step S603 the CPU 310 compares the calculated measured value of CCL17 with the threshold value corresponding to CCL17 stored in the hard disk 313. When the measured value of CCL17 is less than the threshold value, the process proceeds to step S604.
  • step S604 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • step S603 when the measured value of CCL17 is greater than or equal to the threshold value, the process proceeds to step S605.
  • step S605 the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • step S606 the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these three biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these three biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these four biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these four biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for five biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for five biomarkers.
  • the CPU 310 calculates a measured value of each biomarker from the acquired optical information.
  • the CPU 310 stores the measured value of each biomarker in the hard disk 313.
  • the CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • the method for treating a respiratory infection of the present invention includes measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, predicting exacerbation of respiratory infection based on a measured value of the biomarker, and performing medical intervention on a subject predicted to exacerbate respiratory infection in the predicting.
  • medical intervention include drug administration, surgery, immunotherapy, gene therapy, oxygenation procedures, heart-lung machine procedures, and the like.
  • the drug can be appropriately selected from known therapeutic drugs for respiratory infections or candidate medicines therefor.
  • examples of the drug include drugs having an antiviral action, drugs that reduce inflammation, ACE inhibitors, and the like.
  • Specific examples of the drug include favipiravir, lopinavir, ritonavir, nafamostat, camostat, remdesivir, ribavirin, ivermectin, ciclesonide, chloroquine, hydroxychloroquine, interferon, tocilizumab, sarilumab, tofasitinib, baricitinib, ruxolitinib, acalabrutinib, ravulizumab, eritoran, ibudilast, HLCM051, LY3127804, and the like.
  • HISCL refers to a registered trademark of Sysmex Corporation.
  • Serum obtained from 8 patients whose SARS-CoV-2 infection was confirmed by PCR test was used as a biological sample.
  • the serum was prepared from blood collected at a plurality of time point from the day the patient was hospitalized. Information on each patient is shown in Table 1.
  • "onset date” indicates the day when cold symptoms such as fever and cough appeared.
  • "Severity” indicates a final medical condition of each patient after hospitalization.
  • "Mild” indicates a case without pneumonia.
  • Mode indicates a case with pneumonia without oxygen demand.
  • severe indicates a case with pneumonia with oxygen demand.
  • “Critical” indicates a case requiring intensive care management including mechanical ventilation management.
  • Concentration of INF ⁇ 3 as a cytokine not included in measurement items of the above kits was measured by a fully automatic immunoassay device HISCL-5000 (Sysmex Corporation) using the following R1 to R5 reagents.
  • An R1 reagent capture antibody reagent
  • An R1 reagent was prepared by labeling an anti-hIL-28B antibody (clone name: Hyb-TA2650B) provided by the National Center for Global Health and Medicine with biotin by a conventional method, and dissolving the labeled antibody in 1% bovine serum albumin (BSA) and a 0.5% casein-containing buffer.
  • BSA bovine serum albumin
  • R2 reagent solid phase
  • a HISCL (registered trademark) R2 reagent Sysmex Corporation
  • An R3 reagent detection antibody reagent
  • An anti-rhIL-28B antibody (clone name: Hyb-TA2664) provided by the National Center for Global Health and Medicine into a Fab' fragment by a conventional method, labeling this Fab' fragment with ALP by a conventional method, and dissolving the labeled antibody in 1% BSA and a 0.5% casein-containing buffer.
  • R4 reagent measure buffer solution
  • a HISCL R4 reagent Sysmex Corporation
  • R5 reagent As an R5 reagent (ALP substrate solution), a HISCL R5 reagent (Sysmex Corporation) was used. A method for preparing an anti-hIL-28B antibody and an anti-rhIL-28B antibody is described in Japanese Examined Patent No. 6081699 .
  • a measurement procedure according to HISCL-5000 was as follows. After mixing serum (30 ⁇ L) and the R1 reagent (100 ⁇ L), the R2 reagent (30 ⁇ L) was added thereto. The magnetic particles in the obtained mixed solution were magnetically collected to remove the supernatant, and a HISCL washing solution (300 ⁇ L) was added to wash the magnetic particles. The supernatant was removed, and the R3 reagent (100 ⁇ L) was added to the magnetic particles and mixed. The magnetic particles in the obtained mixed solution were magnetically collected to remove the supernatant, and a HISCL washing solution (300 ⁇ L) was added to wash the magnetic particles.
  • hIL-28B 10-046 provided by the National Center for Global Health and Medicine was used. The calibrator was measured in the same manner as serum to prepare a calibration curve. The chemiluminescence intensity obtained by the measurement of each serum was applied to the calibration curve to determine the concentration of INF ⁇ 3.
  • Figs. 5 to 10 show graphs plotting measured values of each patient for IFN ⁇ 3,CXCL11, IP-10, IL-6, CXCL9 and CCL17.
  • elapsed days indicates the number of days from the day when the patient was hospitalized (elapsed days 0). Arrows in the figures indicate a time point when the patient was treated with an oxygen inhaling apparatus or a heart-lung machine.
  • IFNL3 means IFN ⁇ 3.
  • CCL17 can be used as a biomarker to find a patient with the possibility of exacerbation of respiratory infection of the subject is low, by using in combination with IFN ⁇ 3, CXCL11, IP-10, IL-6 or CXCL9.
  • Serum obtained from 20 patients whose SARS-CoV-2 infection was confirmed by PCR test was used as a biological sample.
  • the serum was prepared from blood collected at a plurality of time point from the day the patient was hospitalized. Severity of the 20 patients was mild in 2, moderate in 11, severe in 2, and critical in 5.
  • Figs. 11 and 12 show graphs plotting the measured values of 28 patients, including 20 patients in Example 2 and 8 patients in Example 1, for IFN ⁇ 3 and CCL17.
  • Fig. 11A shows IFN ⁇ 3 measured values for critical and severe patients
  • Fig. 11B shows IPN ⁇ 3 measured values for moderate and mild patients.
  • Fig. 12A shows CCL17 measured values for critical and severe patients
  • Fig. 12B shows CCL17 measured values for moderate and mild patients.
  • “Days after hospitalization” indicates the number of days from the day when the patient was hospitalized (0 day).
  • Example 2 Analysis was performed on the measured values of 28 patients, including 20 patients in Example 2 and 8 patients in Example 1. Of the 28 patients, patients with mild or moderate severity were classified as “low risk group” (hereinafter referred to as "L group”), patients with severe or critical severity were classified as “high risk group” (hereinafter referred to as "H group”), and concentrations of IFN ⁇ 3 and CCL17 in each group were plotted. The results are shown in Figs. 13A and 13B . Horizontal lines in the figures indicate first quartile, median and third quartile of the biomarker concentration of each group.
  • L group low risk group
  • H group high risk group
  • the biomarker concentrations of 28 patients were analyzed by ROC, and an optimum cutoff value (threshold value) for distinguishing between the L group and the H group was set.
  • the set cutoff values were used to calculate sensitivity, specificity and area under the curve (AUC) of determination when determining whether or not the possibility of exacerbation of respiratory infection is high.
  • the obtained ROC curves are shown in Figs. 14A and 14B .
  • the cutoff values of IFN ⁇ 3 and CCL17, and the sensitivity, specificity, AUC and p-values of determination using the cutoff values are shown in Table 2.
  • Event-free survival (EFS) after hospitalization was examined by Kaplan-Meier method for two patient groups classified based on the cutoff values.
  • IFN ⁇ 3 and CCL17 are biomarkers that allow prediction of exacerbation of respiratory infection. It was shown from Fig. 15A that the possibility of exacerbation of respiratory infection is high when the measured value of IFN ⁇ 3 is greater than the cutoff value, and the possibility of exacerbation of respiratory infection is low when the measured value of IFN ⁇ 3 is less than or equal to the cutoff value. It was shown from Fig. 15B that the possibility of exacerbation of respiratory infection is high when the measured value of CCL17 is less than the cutoff value, and the possibility of exacerbation of respiratory infection is low when the measured value of CCL17 is greater than or equal to the cutoff value.

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Abstract

Disclosed is a method for assisting prediction of exacerbation of respiratory infection, comprising measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and a measured value of the biomarker is used as an index to predict exacerbation of the respiratory infection.

Description

    FIELD OF THE INVENTION
  • The present invention relates to a method for assisting exacerbation of respiratory infection. The present invention relates to a reagent kit used for these methods.
    • BACKGROUND
  • Patients with respiratory infections are often mild and do not require hospitalization, but some patients become severe and need to be hospitalized for treatment. Since it is necessary to give priority to treatment of severely ill patients, there is a great need for a means to predict exacerbation of respiratory infection.
  • In view of the above circumstances, the present inventors have attempted to search for a biomarker that can predict exacerbation. An object of the present invention is to provide a new means for predicting exacerbation of respiratory infection by using such biomarker.
  • Japanese Examined Patent No. 6081699 describes that IL (Interleukin)-28B (also called IPNλ3), a type of cytokine, in serum of patient with chronic hepatitis C was detected by a method that specifically measures IL-28B. However, Japanese Examined Patent No. 6081699 does not describe that IPNλ3 was measured in order to predict exacerbation of respiratory infection.
    • SUMMARY OF THE INVENTION
  • The present inventors have found that IFNλ3 (Interferonλ3), CCL17 (CC chemokine ligand 17), CXCL11 (CXC chemokine ligand 11), IP-10 (Interferon-inducible Protein-10), IL-6 and CXCL9 (CXC chemokine ligand 9) can be used as biomarkers for predicting exacerbation of respiratory infection, thereby completing the invention.
  • The present invention provides a method for assisting prediction of exacerbation of respiratory infection, comprising: measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and a measured value of the biomarker is an index to predict exacerbation of the respiratory infection.
  • In a certain embodiment, the biomarker is IFNλ3, CXCL11, IP-10, IL-6 or CXCL9, and
    when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • In a certain embodiment, when the measured value of the biomarker is less than the threshold value corresponding to the biomarker, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a certain embodiment, the biomarker is CCL17, and
    when the measured value of CCL17 is less than a threshold value corresponding to CCL17, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • In a certain embodiment, when the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a certain embodiment, the index is a temporal change in the measured value of the biomarker in the subject.
  • In a certain embodiment, the specimen is whole blood, plasma or serum.
  • In a certain embodiment, the respiratory infection is an infection caused by a virus.
  • In a certain embodiment, the virus is coronavirus or influenzavirus.
  • In a certain embodiment, the virus is α-coronavirus, β-coronavirus, γ-coronavirus or δ-coronavirus.
  • In a certain embodiment, the β-coronavirus is any one of SARS-CoV-2, HCoV-OC43, HCoV-HKU1, SARS-CoV, Bat SL-CoV-WIV1, BtCoV-HKU4, BtCoV-HKU5, MERS-CoV and BtCoV-HKU9.
  • The present invention provides a method for monitoring a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, comprising: acquiring measured values of the biomarker using specimens collected from the subject at a plurality of time points, wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and the measured values are indices to predict exacerbation of the respiratory infection.
  • In a certain embodiment, the biomarker is IFNλ3, CXCL11, IP-10, IL-6 or CXCL9, and
    when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • In a certain embodiment, when the measured value of the biomarker is less than the threshold value corresponding to the biomarker at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a certain embodiment, the biomarker is CCL17, and
    when the measured value of CCL17 is less than a threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  • In a certain embodiment, when the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • The present invention provides a use of a reagent kit for the method according to any one of claims 1 to 15, the reagent kit comprising a reagent comprising a substance capable of specifically binding to the biomarker.
  • Another aspect of the present invention is a method for assisting prediction of exacerbation of respiratory infection, comprising:
    • measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, and
    • predicting exacerbation of respiratory infection based on a measured value of the biomarker,
    • wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • Another aspect of the present invention is a device to assist in predicting exacerbation of respiratory infection, comprising a computer comprising a processor and a memory under control of the processor, in which a computer program for making the computer executes calculating a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, and the output of the measured value of the biomarker is recorded in the memory, wherein the measured value of the biomarker is an index to predict exacerbation of the respiratory infection, and the biomarker comprises at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • Another aspect of the present invention is a computer program to assist in predicting exacerbation of respiratory infection. This computer program is recorded on a computer-readable medium. This computer program makes a computer execute calculating a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, and outputting the measured value of the biomarker. The biomarker comprises at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9.
  • According to the present invention, it is possible to assist in predicting exacerbation of respiratory infection of a subject.
    • BRIEF DESCRIPTION OF THE DRAWINGS
    • Fig. 1A is a schematic diagram showing an example of the reagent kit according to an embodiment of the present invention.
    • Fig. 1B is a schematic diagram showing an example of the reagent kit according to an embodiment of the present invention.
    • Fig. 1C is a schematic diagram showing an example of the reagent kit according to an embodiment of the present invention.
    • Fig. ID is a schematic diagram showing an example of the reagent kit according to an embodiment of the present invention.
    • Fig. 2 is a schematic diagram showing an example of the device according to an embodiment of the present invention.
    • Fig. 3 is a block diagram showing a hardware configuration according to an embodiment of the device of the present invention.
    • Fig. 4A is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 4B is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 4C is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 4D is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 4E is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 4F is a flowchart showing a processing procedure by the device according to an embodiment of the present invention.
    • Fig. 5A is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 5B is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 5C is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 5D is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 5E is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 5F is a graph showing transition of IFNλ3 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 5G is a graph showing transition of IPNλ3 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 5H is a graph showing transition of IPNλ3 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 6A is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 6B is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 6C is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 6D is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 6E is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 6F is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 6G is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 6H is a graph showing transition of CXCL11 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 7A is a graph showing transition of IP-10 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 7B is a graph showing transition of IP-10 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 7C is a graph showing transition of IP-10 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 7D is a graph showing transition of IP-10 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 7E is a graph showing transition of IP-10 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 7F is a graph showing transition of IP-10 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 7G is a graph showing transition of IP-10 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 7H is a graph showing transition of IP-10 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 8A is a graph showing transition of IL-6 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 8B is a graph showing transition of IL-6 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 8C is a graph showing transition of IL-6 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 8D is a graph showing transition of IL-6 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 8E is a graph showing transition of IL-6 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 8F is a graph showing transition of IL-6 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 8G is a graph showing transition of IL-6 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 8H is a graph showing transition of IL-6 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 9A is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 9B is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 9C is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 9D is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 9E is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 9F is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 9G is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 9H is a graph showing transition of CXCL9 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 10A is a graph showing transition of CCL17 concentration in serum of a patient in case No. 7 according to an embodiment of the present invention.
    • Fig. 10B is a graph showing transition of CCL17 concentration in serum of a patient in case No. 11 according to an embodiment of the present invention.
    • Fig. 10C is a graph showing transition of CCL17 concentration in serum of a patient in case No. 5 according to an embodiment of the present invention.
    • Fig. 10D is a graph showing transition of CCL17 concentration in serum of a patient in case No. 9 according to an embodiment of the present invention.
    • Fig. 10E is a graph showing transition of CCL17 concentration in serum of a patient in case No. 12 according to an embodiment of the present invention.
    • Fig. 10F is a graph showing transition of CCL17 concentration in serum of a patient in case No. 4 according to an embodiment of the present invention.
    • Fig. 10G is a graph showing transition of CCL17 concentration in serum of a patient in case No. 8 according to an embodiment of the present invention.
    • Fig. 10H is a graph showing transition of CCL17 concentration in serum of a patient in case No. 10 according to an embodiment of the present invention.
    • Fig. 11A is a graph showing transition of IFNλ3 concentrations in sera of patients in group H according to an embodiment of the present invention.
    • Fig. 11B is a graph showing transition of IFNλ3 concentrations in sera of patients in group L according to an embodiment of the present invention.
    • Fig. 12A is a graph showing transition of CCL17 concentrations in sera of patients in group H according to an embodiment of the present invention.
    • Fig. 12B is a graph showing transition of CCL17 concentrations in sera of patients in group L according to an embodiment of the present invention.
    • Fig. 13A is a graph plotting IFNλ3 concentrations in sera of patients in groups H and L according to an embodiment of the present invention.
    • Fig. 13B is a graph plotting CCL17 concentrations in sera of patients in groups H and L according to an embodiment of the present invention.
    • Fig. 14A is a ROC (receiver operating characteristic) curve when determining exacerbation of respiratory infection based on IFNλ3 concentration according to an embodiment of the present invention.
    • Fig. 14B is a ROC curve when determining exacerbation of respiratory infection based on CCL17 concentration according to an embodiment of the present invention.
    • Fig. 15A is a Kaplan-Meier curve showing a relationship between period after hospitalization and event-free survival rate in a group of patients with IFNλ3 concentration greater than or equal to a cutoff value and in a group of patients with IFNλ3 concentration less than the cutoff value.
    • Fig. 15B is a Kaplan-Meier curve showing a relationship between period after hospitalization and event-free survival rate in a group of patients with CCL17 concentration greater than a cutoff value and in a group of patients with CCL17 concentration less than or equal to the cutoff value.
    DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • In the method for assisting prediction of exacerbation of respiratory infection in a subject of the present invention (hereinafter, also referred to as "prediction method"), first, as a biomarker in a specimen collected from said subject, at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9 is measured. In the prediction method of the present invention, the measured value of the biomarker serves as an index to predict exacerbation of the respiratory infection in said subject.
  • Respiratory infection refers to a disease caused by infection of respiratory organ such as nasal cavity, pharynx, trachea, bronchi or alveoli with a pathogen. The pathogen is not particularly limited, and examples thereof include viruses, bacteria, fungi, parasites and the like. Examples of the virus include coronavirus, influenza virus, and the like. The coronavirus is not particularly limited, and examples thereof include α-coronavirus, β-coronavirus, γ-coronavirus, and δ-coronavirus. Examples of the β-coronavirus include SARS-CoV-2, SARS-CoV, HCoV-OC43, HCoV-HKU1, Bat SL-CoV-WIV1, BtCoV-HKU4, BtCoV-HKU5, MERS-CoV, BtCoV-HKU9, and the like.
  • Examples of the subject in the methods of the present invention include a patient suffering from a respiratory infection and a person suspected of having a respiratory infection. The wording "patient suffering from a respiratory infection" refers to a person whose respiratory infection has been confirmed due to detection of a pathogen or the like and has not yet become severe. The person suspected of having a respiratory infection include, for example, those who have cold symptoms such as fever, cough, runny nose and sore throat and/or symptoms specific to prescribed respiratory infections such as feeling of dyspnea, short breath during exertion and abnormal taste and smell, and those having come into contact with and those suspected of having come into contact with patient with respiratory infection. Contact with patient with respiratory infection refers to, for example, an act such as talking with the patient within a distance of 1 m, staying in a closed space where the patient is present, and being contacted with saliva, coughing or the like of the patient.
  • In particular embodiments, the reference to "exacerbation of respiratory infection" refers to developing pneumonia requiring oxygen inhalation or being in a condition requiring intensive care management including mechanical ventilation management.
  • The specimen used in the methods of the invention is not particularly limited as long as it is a liquid sample collected from a subject and suspected of containing the biomarker. Examples of such liquid sample include blood samples, cerebrospinal fluid, sputum, bronchoalveolar lavage fluid, nasopharyngeal swab, lymph fluid, urine, stool, saliva, and the like. Among them, blood samples are preferred. Examples of the blood sample include whole blood, plasma and serum, and plasma and serum are particularly preferable.
  • When insoluble contaminants such as cells are contained in the specimen, for example, impurities may be removed from the specimen by a known means such as centrifugal separation and filtration. The specimen may be diluted with an appropriate aqueous medium as necessary. Such an aqueous medium is not particularly limited as long as it does not interfere with the measurement described herein below, and examples thereof include water, physiological saline, a buffer solution, and the like. The buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutrality (for example, a pH of 6 or more and 8 or less). Examples of the buffer solution include Good buffers such as HEPES, MES, Tris and PIPES, phosphate buffered saline (PBS), and the like.
  • The biomarker measured by the prediction method of the present invention is one or more protein molecules selected from IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9. IFNλ3, also called IL-28B, is a protein consisting of 200 amino acids encoded by a gene of about 1.5 Kb located on chromosome 19. IFNλ3 has 25 signal peptides at N-terminus, and the signal peptide is cleaved when IPNλ3 is secreted extracellularly. CCL17 is also called TARC (Thymus- and activation-regulated chemokine) and is a type of Th2 type chemokine. CXCL11 is a chemokine also called I-TAC (Interferon-inducible T-cell Chemoattractant), and is a ligand for CXCR3 receptor. IP-10, also called CXCL10, is a type of Thl type chemokine. IL-6 is a type of TH2 type cytokine. CXCL9, also called MIG (Monokine induced by interferon γ), is a ligand for CXCR3 receptor and is a type of Thl type chemokine as well as IP-10. These protein molecules themselves are known, and their amino acid sequences can be acquired from known databases such as NCBI (National Center for Biotechnology Information). For example, IFNλ3 may have an amino acid sequence represented by SEQ ID NO: 1 or may have an amino acid sequence represented by SEQ ID NO: 2.
  • In the particular embodiments, it is preferable to acquire measured values of two or more biomarkers selected from IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9 from the viewpoint of improving accuracy of prediction of exacerbation. Examples of the two or more biomarkers include one of the following combinations:
    • a combination of IFNλ3 and at least one selected from the group consisting of CCL17, CXCL11, IP-10, IL-6 and CXCL9;
    • a combination of CCL17 and at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9;
    • a combination of CXCL11 and at least one selected from the group consisting of IFNλ3, CCL17, IP-10, IL-6 and CXCL9;
    • a combination of IP-10 and at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IL-6 and CXCL9;
    • a combination of IL-6 and at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, and CXCL9; and
    • a combination of CXCL9 and at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10 and IL-6.
  • In the particular embodiments, it is particularly preferable to acquire a measured value of IFNλ3 and a measured value of CCL17.
  • Any known method can be used for measuring the biomarker, and the method is not particularly limited. In the particular embodiments, a method of capturing a biomarker using a substance capable of specifically binding to the biomarker is preferable. The biomarker contained in the specimen can be measured by detecting the biomarker captured by such a substance by a method known in the art. The measured value of the biomarker may be a value that reflects an amount or concentration in the specimen. The measured value may be a concentration or a value reflecting the concentration calculated based on a measurement result of a calibrator. The "value reflecting the concentration" depends on a type of labeling substance described herein below, and examples thereof include a measured value of fluorescence intensity, a measured value of emission intensity, a measured value of radioactivity, and the like.
  • Examples of the substance capable of specifically binding to the biomarker include an antibody, an aptamer and the like, among which an antibody is particularly preferable. An antibody against the biomarker is not particularly limited as long as it is an antibody capable of specifically binding to the biomarker. Such an antibody may be any of monoclonal antibodies, polyclonal antibodies, and fragments thereof (for example, Fab, F(ab')2, etc.). A commercially available antibody may be used.
  • As an antibody capable of specifically binding to IFNλ3, for example, a monoclonal antibody having a heavy chain variable region domain and a light chain variable region domain described in any one of (1) to (5) below or a fragment thereof may be used.
    1. (1) A heavy chain variable region domain including a heavy chain complementarity determining region 1 including amino acid sequences 31 to 35, a heavy chain complementarity determining region 2 including amino acid sequences 50 to 66, and a heavy chain complementarity determining region 3 including amino acid sequences 99 to 113 in an amino acid sequence represented by SEQ ID NO: 3, and a light chain variable region domain including a light chain complementarity determining region 1 including amino acid sequences 24 to 38, a light chain complementarity determining region 2 including amino acid sequences 54 to 60, and a light chain complementarity determining region 3 including amino acid sequences 93 to 101 in an amino acid sequence represented by SEQ ID NO: 4;
    2. (2) A heavy chain variable region domain including a heavy chain complementarity determining region 1 including amino acid sequences 31 to 35, a heavy chain complementarity determining region 2 including amino acid sequences 50 to 66, and a heavy chain complementarity determining region 3 including amino acid sequences 99 to 109 in an amino acid sequence represented by SEQ ID NO: 5, and a light chain variable region domain including a light chain complementarity determining region 1 including amino acid sequences 24 to 38, a light chain complementarity determining region 2 including amino acid sequences 54 to 60, and a light chain complementarity determining region 3 including amino acid sequences 93 to 101 in an amino acid sequence represented by SEQ ID NO: 6;
    3. (3) A heavy chain variable region domain including a heavy chain complementarity determining region 1 including amino acid sequences 31 to 35, a heavy chain complementarity determining region 2 including amino acid sequences 50 to 66, and a heavy chain complementarity determining region 3 including amino acid sequences 99 to 106 in an amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region domain including a light chain complementarity determining region 1 including amino acid sequences 24 to 38, a light chain complementarity determining region 2 including amino acid sequences 54 to 60, and a light chain complementarity determining region 3 including amino acid sequences 93 to 101 in an amino acid sequence represented by SEQ ID NO: 8;
    4. (4) A heavy chain variable region domain including a heavy chain complementarity determining region 1 including amino acid sequences 31 to 35, a heavy chain complementarity determining region 2 including amino acid sequences 50 to 66, and a heavy chain complementarity determining region 3 including amino acid sequences 99 to 106 in an amino acid sequence represented by SEQ ID NO: 9, and a light chain variable region domain including a light chain complementarity determining region 1 including amino acid sequences 24 to 38, a light chain complementarity determining region 2 including amino acid sequences 54 to 60, and a light chain complementarity determining region 3 including amino acid sequences 93 to 101 in an amino acid sequence represented by SEQ ID NO: 10; or
    5. (5) A heavy chain variable region domain including a heavy chain complementarity determining region 1 including amino acid sequences 31 to 35, a heavy chain complementarity determining region 2 including amino acid sequences 50 to 66, and a heavy chain complementarity determining region 3 including amino acid sequences 99 to 106 in an amino acid sequence represented by SEQ ID NO: 11, and a light chain variable region domain including a light chain complementarity determining region 1 including amino acid sequences 24 to 38, a light chain complementarity determining region 2 including amino acid sequences 54 to 60, and a light chain complementarity determining region 3 including amino acid sequences 93 to 101 in an amino acid sequence represented by SEQ ID NO: 12.
  • The method used for measuring a biomarker using an antibody is not particularly limited and can be appropriately selected from known immunoassays. In the particular embodiments, an enzyme-linked immunosorbent assay (ELISA method) is preferred, and a sandwich ELISA method being particularly preferred. As an example of the measurement step, the case of measuring a biomarker in the specimen by a sandwich ELISA method will be described below.
  • First, a complex containing a biomarker, an antibody for capturing the biomarker (hereinafter also referred to as "capture antibody") and an antibody for detecting the biomarker (hereinafter also referred to as "detection antibody") is formed and/or captured on a solid phase. When the specimen contains a biomarker, a complex can be formed by mixing the specimen, a capture antibody, and a detection antibody. Then, a solution containing the complex is brought into contact with a solid phase capable of immobilizing the capture antibody, whereby the complex can be captured on the solid phase. Alternatively, a solid phase preliminarily immobilized with the capture antibody may be used. That is, a solid phase immobilized with the capture antibody, the specimen, and the detection antibody are brought into contact with each other, whereby the complex can be formed on the solid phase. When both the capture antibody and the detection antibody are monoclonal antibodies, it is preferable that the epitopes be different from each other.
  • The mode of immobilization of the capture antibody on the solid phase is not particularly limited. For example, the capture antibody and the solid phase may be bound directly, or the capture antibody and the solid phase may be indirectly bound via another substance. Examples of the direct binding include physical adsorption and the like. Examples of the indirect bond include a bond via a combination of biotins and avidins. In this case, by preliminarily modifying the capture antibody with biotins and previously binding avidins to the solid phase, the capture antibody and the solid phase can be indirectly bound via the bond between the biotins and the avidins. The biotins include biotin and biotin analogs such as desthiobiotin. The avidins include avidin and analogs of avidins such as streptavidin and tamavidin (registered trademark).
  • The material of the solid phase is not particularly limited. For example, the material can be selected from organic polymer compounds, inorganic compounds, biopolymers, and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene, and the like. Examples of the inorganic compound include magnetic bodies (iron oxide, chromium oxide, ferrite, and the like), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose, and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, test tubes, and the like. Among them, particles are preferable, and magnetic particles are particularly preferable.
  • In particular embodiments, B/F (Bound/Free) separation for removing an unreacted free component not forming a complex may be performed between the process of forming the complex and the process of detecting the complex. The unreacted free component refers to a component not constituting a complex. Examples thereof include capture antibodies not bound to the biomarker, detection antibodies, and the like. The means of B/F separation is not particularly limited, and when the solid phase is a particle, B/F separation can be performed by recovering only the solid phase capturing the complex by centrifugation. When the solid phase is a container such as a microplate or a microtube, B/F separation can be performed by removing a liquid containing an unreacted free component. When the solid phase is a magnetic particle, B/F separation can be performed by aspirating and removing a liquid containing an unreacted free component by a nozzle while magnetically constraining the magnetic particles with a magnet, which is preferable from the viewpoint of automation. After removing the unreacted free component, the solid phase capturing the complex may be washed with a suitable aqueous medium such as PBS.
  • Moreover, a measured value of a biomarker contained in the specimen can be acquired by detecting the complex formed on the solid phase by a method known in the art. For example, when an antibody labeled with a labeling substance is used as a detection antibody, the measured value of the marker in the liquid sample can be acquired by detecting a signal generated by the labeling substance. Alternatively, also when a labeled secondary antibody against the detection antibody is used, the measured value of the biomarker in the liquid sample can be acquired in the same manner.
  • As an example of a method for measuring a biomarker using an antibody, the immune complex transfer method described in Japanese Laid-Open Patent Publication No. H01-254868 can be also used.
  • The phrase "detecting a signal" herein includes qualitatively detecting the presence or absence of a signal, quantifying a signal intensity, and semi-quantitatively detecting the intensity of a signal. Semi-quantitative detection means to show the intensity of the signal in stages like "no signal generated", "weak", "medium", "strong", and the like. In the present embodiment, it is preferable to detect the intensity of a signal quantitatively or semi-quantitatively.
  • The labeling substance is not particularly limited. For example, the labeling substance may be a substance which itself generates a signal (hereinafter also referred to as "signal generating substance") or a substance which catalyzes the reaction of other substances to generate a signal. Examples of the signal generating substance include fluorescent substances, radioactive isotopes, and the like. Examples of the substance that catalyzes the reaction of other substances to generate a detectable signal include enzymes. Examples of the enzymes include alkaline phosphatase, peroxidase, β-galactosidase, luciferase, and the like. Examples of the fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), fluorescent proteins such as GFP, and the like. Examples of the radioactive isotopes include 125I, 14C, 32P, and the like. Among them, an enzyme is preferable as a labeling substance, and alkaline phosphatase and peroxidase are particularly preferable.
  • Methods for detecting a signal themselves are known in the art. In particular embodiments, a measurement method according to the type of signal derived from the labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting a substrate for the enzyme can be measured by using a known apparatus such as a spectrophotometer.
  • The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase (ALP) is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (disodium 4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate) and CSPD (registered trademark) (disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate, and p-nitrophenyl phosphate. When peroxidase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as luminol and derivatives thereof, and chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB).
  • When the labeling substance is a radioactive isotope, radiation as a signal can be measured using a known apparatus such as a scintillation counter. When the labeling substance is a fluorescent substance, fluorescence as a signal can be measured using a known apparatus such as a fluorescence microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.
  • The detection result of the signal can be used as the measured value of the biomarker. For example, when quantitatively detecting the intensity of a signal, the signal intensity value itself or a value acquired from the measured value can be used as the measured value of the biomarker. Examples of the value acquired from the measured value of the signal intensity include a value acquired by subtracting the measured value of a negative control sample or the background value from the measured value, a value acquired by applying the measured value to a calibration curve, and the like. The negative control sample can be appropriately selected, and examples thereof include a specimen obtained from a mildly ill patient (for example, an infectious disease patient who has recovered without becoming severe), a specimen obtained from a healthy person, and the like.
  • In particular embodiments, it is preferable to measure the biomarker contained in the specimen by an immunoassay such as an EIA method or an ELISA method. Biomarkers can be measured using commercially available devices and reagents such as the HISCL series (manufactured by Sysmex Corporation) and the Bio-Plex Multiplex System (manufactured by Bio-Rad Laboratories, Inc.).
  • In particular embodiments, the measured values of the biomarkers described above can be used as an index indicating whether or not a respiratory infection of a subject is exacerbated or likely to exacerbate. For example, by comparing the acquired measured value of the biomarker with a threshold value corresponding to the biomarker (i.e. a threshold value previously established for the biomarker), the measured value of the biomarker may be used as an index suggesting that the possibility or likeliness of exacerbation of respiratory infection of the subject is high or low. In particular embodiments, the possibility or likeliness of exacerbation of the respiratory infection is a risk of exacerbation of the respiratory infection of the subject after a lapse of a predetermined period or within a predetermined period (for example, 1 day to 1 month) from a date when the specimen was collected from the subject.
  • As shown in the examples described later, IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 showed high values in a patient with severe respiratory infection and low values in a patient with mild respiratory infection. Measured values of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 can be used as indexes suggesting the possibility or likeliness of exacerbation of respiratory infection, by comparison with threshold values corresponding to each biomarker (i.e. for each biomarker). In one embodiment, when the biomarker contains IFNλ3 and the measured value of IFNλ3 is greater than or equal to a threshold value corresponding to IFNλ3, the possibility or likeliness of exacerbation of respiratory infection in the subject is suggested to be high. When the measured value of IFNλ3 is less than the threshold value corresponding to IFNλ3, the possibility or likeliness of exacerbation of respiratory infection in the subject is suggested to be low.
  • In one embodiment, when the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to a threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of CXCL11 is less than the threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In one embodiment, when the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to a threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of IP-10 is less than the threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In one embodiment, when the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to a threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of IL-6 is less than the threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In one embodiment, when the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to a threshold value corresponding to CXCL9, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high, and when the measured value of CXCL9 is less than the threshold value corresponding to CXCL9, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In one embodiment, the biomarker contains at least two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be suggested by their measured values. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When all of the measured values of the biomarkers selected from the group are less than threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • As an example, when the biomarkers are two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and either or both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are five consisting of IFNλ3, CXCL11, IP-10, IL-6, and CXCL9 and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • In another embodiment, the biomarker contains at least two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject can be classified into three stages by their measured values. Specifically:
    • when all of the measured values of the biomarkers selected from the group are greater than or equal to the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection is suggested to be high;
    • when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection is suggested to be medium; and
    • when all of the measured values of the biomarkers selected from the group are less than the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection is suggested to be low.
  • As an example, when the biomarkers are two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. In another example, when the biomarkers are five consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high.
  • In particular embodiments, CCL17 in the specimen may be measured. As shown in the examples described herein below, CCL17, unlike other biomarkers, showed a low value in a patient with severe respiratory infection and a high value in a patient with mild respiratory infection. The measured value of CCL17 can be used as an index suggesting the possibility of exacerbation of respiratory infection, by comparison with a threshold value corresponding to CCL17. In one embodiment, when the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a preferred embodiment, CCL17 is used in combination with at least one biomarker selected from IFNλ3, CXCL11, IP-10, IL-6 and CXCL9. For example, the biomarker contains at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be suggested by their measured values. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, and/or the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When all of the measured values of the biomarkers selected from the group are lower than the threshold values corresponding to each biomarker, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In another embodiment, the biomarker contains at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject can be classified into three stages by their measured values. Specifically:
    • when all of the measured values of the biomarkers selected from the group are greater than or equal to the threshold values corresponding to each biomarker, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high;
    • when at least one of the measured values of the biomarkers selected from the group is greater than or equal to the threshold value corresponding to the biomarker, and the measured value of CCL17 is greater than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be medium;
    • when at least one of the measured values of the biomarkers selected from the group is less than the threshold value corresponding to the biomarker, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be medium; and
    • when all of the measured values of the biomarkers selected from the group are lower than the threshold values corresponding to each biomarker, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a more specific example, biomarkers contain IFNλ3 and CCL17,
    • when the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high;
    • when the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be medium;
    • when the measured value of IPNλ3 is less than the threshold value corresponding to IFNλ3, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be medium; and
    • when the measured value of IPNλ3 is less than the threshold value corresponding to IFNλ3, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • The threshold values corresponding to each biomarker are not particularly limited and can be set as appropriate. For example, specimens are collected from a plurality of patients with respiratory infection, and biomarkers in the specimens are measured to obtain measured values. After a predetermined period (for example, 2 weeks) has passed since the specimens were collected, whether or not the respiratory infection has become severe in these subjects is confirmed. The acquired measured value data is classified into data of a group of severely ill patients and data of a group of non-severely ill patients. Then, for each biomarker, a value that can most accurately distinguish between the group of severely ill patients and the group of non-severely ill patients is determined, and the value is set as a threshold value. In setting the threshold value, it is possible to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.
  • In particular embodiments, the threshold value corresponding to IFNλ3 (or for IFNλ3) is set in the range of, for example, 4 pg/mL or more and 15 pg/mL or less, i.e. the threshold is a value between 4 and 15 pg/mL. In particular embodiments, the threshold value corresponding to CXCL11 is set in the range of, for example, 20 pg/mL or more and 40 pg/mL or less, i.e. the threshold is a value between 20 and 40 pg/mL. In particular embodiments, the threshold value corresponding to IP-10 is set in the range of, for example, 400 pg/mL or more and 1200 pg/mL or less, i.e. the threshold is a value between 400 and 1200 pg/mL. In particular embodiments, the threshold value corresponding to IL-6 is set in the range of, for example, 4 pg/mL or more and 6 pg/mL or less, i.e. the threshold is a value between 4 and 6 mL. In particular embodiments, the threshold value corresponding to CXCL9 is set in the range of, for example, 30 pg/mL or more and 40 pg/mL or less, i.e. the threshold is a value between 30 and 40 pg/mL. In particular embodiments, the threshold value corresponding to CCL17 is set in the range of, for example, 40 pg/mL or more and 100 pg/mL or less, i.e. the threshold is a value between 40 and 100 pg/mL.
  • Healthcare professionals such as doctors may combine the suggestion from the measured value of the biomarker with other information to determine the risk of exacerbation of respiratory infection. The "other information" includes findings on X-ray or CT images of the lungs and other medical findings.
  • When the biomarker is used as an index, this may refer to the absolute value of the biomarker in the sample of the patient. Additionally or alternatively, as an index to predict exacerbation of the respiratory infection of the subject in particular embodiments, temporal change of the measured value of the biomarker in the subject may be acquired. The temporal change of the measured value of the biomarker is not particularly limited as long as it is information showing transition of the measured value of the biomarker in the specimen collected from the subject a plurality of times periodically or irregularly over time. Examples of such temporal change include values calculated from a plurality of measured values (for example, the difference, ratio, etc. of the measured values of two specimens collected at any two time points), records of the measured values (for example, a table of measured values, a graph plotting measured values, etc.), and the like. In particular embodiments, the methods of the present invention involve determining the biomarker in two or more specimens collected from the subject at different time points and determining the temporal change of the value of said biomarker. In particular embodiments, the methods of the present invention involve determining the biomarker in two or more specimens collected from the subject at different time points and determining the likeliness of exacerbation based on said two or more measurements, as detailed for the method of monitoring described below.
  • In particular embodiments, when the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high based on the measured value of the biomarker, it is possible to perform medical intervention to prevent the exacerbation of respiratory infection on the subject. Examples of the medical intervention include drug administration, surgery, immunotherapy, gene therapy, oxygenation procedures, heart-lung machine procedures, and the like. The drug can be appropriately selected from known therapeutic drugs for respiratory infections or candidate medicines therefor. For example, when the respiratory infection is SARS-CoV-2 infection, examples of the drug include drugs having an antiviral action, drugs that reduce inflammation, ACE inhibitors, and the like. Specific examples of the drug include favipiravir, lopinavir, ritonavir, nafamostat, camostat, remdesivir, ribavirin, ivermectin, ciclesonide, chloroquine, hydroxychloroquine, interferon, tocilizumab, sarilumab, tofasitinib, baricitinib, ruxolitinib, acalabrutinib, ravulizumab, eritoran, ibudilast, HLCM051, LY3127804, and the like.
  • The prediction method of the present invention may include predicting exacerbation of respiratory infection, based on a measured value of a biomarker determined in the specimen collected from the subject. In the predicting, for example, the measured value of the biomarker is compared with a threshold value corresponding to the biomarker, and based on the comparison result, it may be determined whether the possibility or likeliness of exacerbation of respiratory infection of the subject is high or low. Details of the threshold value corresponding to the biomarker are as described above.
  • In particular embodiments, when the biomarker contains IFNλ3 and the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of IFNλ3 is less than the threshold value corresponding to IFNλ3, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In particular embodiments, when the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to the threshold value corresponding to CXCL11, the possibility of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of CXCL11 is less than the threshold value corresponding to CXCL11, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In one embodiment, when the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to the threshold value corresponding to IP-10, the possibility of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of IP-10 is less than the threshold value corresponding to IP-10, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In one embodiment, when the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to the threshold value corresponding to IL-6, the possibility of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of IL-6 is less than the threshold value corresponding to IL-6, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In one embodiment, when the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to the threshold value corresponding to CXCL9, the possibility of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of CXCL9 is less than the threshold value corresponding to CXCL9, the possibility of exacerbation of respiratory infection of the subject can be determined to be low.
  • In one embodiment, when the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. When the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In particular embodiments, the biomarker contains at least two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be determined based on their measured values. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. When all of the measured values of the biomarkers selected from the group are less than the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • As an example, when the biomarkers are two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and either or both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. In another example, when the biomarkers are three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. In another example, when the biomarkers are four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. In another example, when the biomarkers are five consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and at least one of them is greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high.
  • In another embodiment, the biomarker contains at least two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be determined in three stages based on their measured values. Specifically:
    • when all of the measured values of the biomarkers selected from the group are greater than or equal to the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection is determined to be high;
    • when at least one of the measured values of the biomarkers selected from the group is greater than or equal to the threshold value corresponding to the biomarker, the possibility or likeliness of exacerbation of respiratory infection is determined to be medium; and
    • when all of the measured values of the biomarkers selected from the group are less than the threshold values corresponding to each biomarker, the possibility or likeliness of exacerbation of respiratory infection can be determined to be low.
  • As an example, when the biomarkers are two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and both of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high. In another example, when the biomarkers are three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high. In another example, when the biomarkers are four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high. In another example, when the biomarkers are five consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and all of them are greater than or equal to the corresponding threshold values, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be high.
  • In particular embodiments, the biomarker contains at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be determined based on their measured values in a sample of said subject. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, and/or the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be high. When all of the measured values of the biomarkers selected from the group are lower than the threshold values corresponding to each biomarker, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • In particular embodiments, the biomarker contains at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be determined in three stages based on their measured values. Specifically:
    • when all of the measured values of the biomarkers selected from the group are greater than or equal to the threshold values corresponding to each biomarker, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection in the subject is determined to be high;
    • when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be medium;
    • when at least one of the measured values of the biomarkers selected from the group is less than the threshold value corresponding to the biomarker and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be medium; and
    • when all of the measured values of the biomarkers selected from the group are lower than the threshold values corresponding to each biomarker, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection in the subject can be determined to be low.
  • In a more specific example, biomarkers contain IFNλ3 and CCL17,
    • when the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection in the subject is determined to be high;
    • when the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be medium;
    • when the measured value of IPNλ3 is less than the threshold value corresponding to IFNλ3, and the measured value of CCL17 is less than the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject is determined to be medium; and
    • when the measured value of IFNλ3 is lower than the threshold value corresponding to IFNλ3, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility or likeliness of exacerbation of respiratory infection of the subject can be determined to be low.
  • The application thus also provides methods for monitoring a measured value of a biomarker in a specimen collected from a subject (hereinafter, also referred to as "monitoring method"). These methods are of particular interest in subjects suffering from a respiratory infection and a person suspected of having a respiratory infection and can be used to monitor the risk of exacerbation of respiratory infection of the subject over time. In the monitoring method of the present invention, the measured value of the biomarker is acquired using specimens collected from the subject at a plurality of time points. Details of the subject, the specimen, the biomarker and acquisition of the measured value thereof are the same as those described for the prediction method of the present embodiment described above.
  • In particular embodiments, the plurality of time points may be two or more different time points. For example, the plurality of time points includes a first time point and a second time point different from the first time point. The first time point is not particularly limited and can be any time point. For example, the first time point may be a time point when the subject is found to have a respiratory infection, a time point when the subject develops symptoms of a respiratory infection, a time point when the subject is hospitalized, or the like The second time point is not particularly limited as long as it differs from the first time point. Preferably, the second time point is a time point when a period within one month has passed from the first time point. Specifically, the second time point is a time point when 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 2 weeks, 3 weeks, 4 weeks or one month has passed from the first time point.
  • In particular embodiments, the "specimens collected from the subject at a plurality of time points" are specimens collected from the same subject at each of the plurality of time points. For example, it includes a first specimen collected from a subject at a first time point and a second specimen collected from the subject at a second time point different from the first time point. In the methods of the present embodiment, the biomarker may be measured each time a specimen is collected, or each collected specimen may be stored and measured collectively.
  • In particular embodiments of the methods of the present invention, the measured value of the biomarker in the same subject is monitored and used as an index to predict exacerbation of the respiratory infection. In a preferred embodiment, the measured values of the same biomarker at a plurality of time points are acquired. The measured value of the biomarker measured from each specimen is compared with the threshold value corresponding to the biomarker, and based on the comparison result, the possibility, also referred to as the the risk or likeliness, of exacerbation of respiratory infection of the subject may be suggested to be high or low. Details of the threshold value corresponding to the biomarker are similar to those described for the prediction methods described above.
  • In particular embodiments, when the biomarker contains IFNλ3 and the measured value of IFNλ3 is greater than or equal to the threshold value corresponding to IFNλ3 at at least one of the plurality of time points, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of IFNλ3 is less than the threshold value corresponding to IFNλ3 at any of the plurality of time points, the possibility or likeliness of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, when the biomarker contains CXCL11 and the measured value of CXCL11 is greater than or equal to the threshold value corresponding to CXCL11 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of CXCL11 is less than the threshold value corresponding to CXCL11 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, when the biomarker contains IP-10 and the measured value of IP-10 is greater than or equal to the threshold value corresponding to IP-10 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of IP-10 is less than the threshold value corresponding to IP-10 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, when the biomarker contains IL-6 and the measured value of IL-6 is greater than or equal to the threshold value corresponding to IL-6 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of IL-6 is less than the threshold value corresponding to IL-6 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, when the biomarker contains CXCL9 and the measured value of CXCL9 is greater than or equal to the threshold value corresponding to CXCL9 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of CXCL9 is less than the threshold value corresponding to CXCL9 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, the biomarker contains at least two selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and the exacerbation risk of the respiratory infection of the subject may be suggested by the measured values of the biomarkers at a plurality of time points. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When all of the measured values of the biomarkers selected from the group are less than the threshold values corresponding to each biomarker at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In particular embodiments, the measured value of CCL17 in each of the specimens taken from the subject may be acquired. In one embodiment, when the biomarker contains CCL17 and the measured value of CCL17 is less than the threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • In a further embodiment, the biomarker contains at least one selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9, and CCL17, and the exacerbation risk of the respiratory infection of the subject may be suggested by the measured values of the biomarkers at a plurality of time points. Specifically, when at least one of the measured values of the biomarkers selected from the group is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, and/or the measured value of CCL17 is less than the threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high. When all of the measured values of the biomarkers selected from the group are lower than the threshold values corresponding to each biomarker at any of the plurality of time points, and the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  • The conditions for terminating the monitoring method of the present invention are not particularly limited, and a healthcare professional such as a doctor may appropriately determine the conditions. For example, when the possibility or likeliness or risk of exacerbation of respiratory infection of the subject was suggested to be high by the measured values of the biomarkers acquired from specimens collected from the subject at a plurality of time points, the monitoring method of the present embodiment may be terminated. In this case, it can be decided that it is preferable to perform medical intervention for the exacerbation of respiratory infection on the subject. Details of the medical intervention are as described above. Alternatively, when the possibility of exacerbation of respiratory infection of the subject was suggested to be low by the measured values of the biomarkers acquired from specimens collected from the subject at a plurality of time points, and any symptoms of respiratory infection is not recognized on the subject, the monitoring method of the present embodiment may be terminated. Additionally, or alternatively the methods of the present invention such as the diagnosis or monitoring methods described herein may be used to determine which treatment will be applied.
  • In each of the embodiments described above, when the measured values of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are the same as the threshold values corresponding to each biomarker, it has been stated that the possibility of exacerbation of respiratory infection of the subject is suggested to be high. However, the possibility of exacerbation of respiratory infection of the subject may be suggested to be low. In each of the embodiments described above, when the measured value of CCL17 is the same as the threshold value corresponding to CCL17, it has been stated that the possibility of exacerbation of respiratory infection of the subject is suggested to be low. However, the possibility of exacerbation of respiratory infection of the subject may be suggested to be high.
  • The invention also provides a reagent kit for use in the prediction methods and/or monitoring methods of the present invention described above. The reagent kit of the present invention includes at least one reagent selected from a reagent containing a substance capable of specifically binding to IFNλ3, a reagent containing a substance capable of specifically binding to CCL17, a reagent containing a substance capable of specifically binding to CXCL11, a reagent containing a substance capable of specifically binding to IP-10, a reagent containing a substance capable of specifically binding to IL-6, and a reagent containing a substance capable of specifically binding to CXCL9. In a further embodiment, the reagent kit may include at least one reagent selected from the reagent containing a substance capable of specifically binding to IFNλ3, the reagent containing a substance capable of specifically binding to CXCL11, the reagent containing a substance capable of specifically binding to IP-10, the reagent containing a substance capable of specifically binding to IL-6, and the reagent containing a substance capable of specifically binding to CXCL9, and the reagent containing a substance capable of specifically binding to CCL17. In a preferred embodiment, the reagent kit includes the reagent containing a substance capable of specifically binding to IFNλ3 and the reagent containing a substance capable of specifically binding to CCL17. Examples of the substance capable of specifically binding to each biomarker include an antibody, an aptamer, and the like. The antibody is particularly preferable among them.
  • Fig. 1A shows an example of the reagent kit of the present embodiment. In Fig.1A, 11 denotes a reagent kit, 12 denotes a first container containing a reagent containing a substance capable of specifically binding to IFNλ3, 13 denotes a second container containing a reagent containing a substance capable of specifically binding to CCL17, 14 denotes a packing box, and 15 denotes an attached document. Composition, usage, storage method, etc. of each reagent may be described in the attached document. The reagent kit of this example includes the reagent containing a substance capable of specifically binding to IFNλ3 and the reagent containing a substance capable of specifically binding to CCL17, but in place of these reagents, reagents containing a substance capable of specifically binding to another biomarker may be included.
  • In a preferred embodiment, the reagent kit of the present embodiment includes a capture antibody and a detection antibody for the biomarker. The detection antibody may be labeled with a labeling substance. Details of the capture antibody, the detection antibody and the labeling substance are the same as those described for the prediction method of the present embodiment. The reagent kit may include a solid phase and a substrate. Details of the solid phase and the substrate are the same as those described for the prediction method of the present embodiment.
  • Fig. 1B shows an example of a reagent kit of a further embodiment. In Fig. 1B, 21 denotes a reagent kit, 22 denotes a first container containing a reagent containing a capture antibody for IFNλ3, 23 denotes a second container containing a reagent containing a detection labeled antibody for IFNλ3, 24 denotes a third container containing a reagent containing a capture antibody for CCL17, 25 denotes a fourth container containing a reagent containing a detection labeled antibody for CCL17, 26 denotes an attached document, and 27 denotes a packing box. The reagent kit of this example includes reagents each containing the capture antibody and the detection labeled antibody for IFNλ3 and reagents each containing the capture antibody and the detection labeled antibody for CCL17, but in place of these reagents, reagents each containing a capture antibody for another biomarker and a detection labeled antibody for another biomarker may be included.
  • It is preferable that any of the above reagent kits include a calibrator. Examples of the calibrator include a calibrator for quantifying IFNλ3 (calibrator for IFNλ3), a calibrator for quantifying CXCL11 (calibrator for CXCL11), a calibrator for quantifying IP-10 (calibrator for IP-10), a calibrator for quantifying IL-6 (calibrator for IL-6), a calibrator for quantifying CXCL9 (calibrator for CXCL9), and a calibrator for quantifying CCL17 (calibrator for CCL17). The calibrator for IFNλ3 may include, for example, a buffer solution containing no IFNλ3 (negative control) and a buffer solution containing IFNλ3 at a known concentration. The calibrator for CXCL11 may include, for example, a buffer solution containing no CXCL11 (negative control) and a buffer solution containing CXCL11 at a known concentration. The calibrator for IP-10 may include, for example, a buffer solution containing no IP-10 (negative control) and a buffer solution containing IP-10 at a known concentration. The calibrator for IL-6 may include, for example, a buffer solution containing no IL-6 (negative control) and a buffer solution containing IL-6 at a known concentration. The calibrator for CXCL9 may include, for example, a buffer solution containing no CXCL9 (negative control) and a buffer solution containing CXCL9 at a known concentration. The calibrator for CCL17 may include, for example, a buffer solution containing no CCL17 (negative control) and a buffer solution containing CCL17 at a known concentration.
  • Fig. 1C shows an example of a reagent kit of a further embodiment. In Fig. 1C, 31 denotes a reagent kit, 32 denotes a first container containing a reagent containing a capture antibody for IFNλ3, 33 denotes a second container containing a reagent containing a detection labeled antibody for IFNλ3, 34 denotes a third container containing a reagent containing a capture antibody for CCL17, 35 denotes a fourth container containing a reagent containing a detection labeled antibody for CCL17, 36 denotes a fifth container containing a buffer solution containing neither IFNλ3 nor CCL17, 37 denotes a sixth container containing a buffer solution containing IFNλ3 and CCL17 at each predetermined concentrations, 38 denotes a packing box, and 39 denotes an attached document. A buffer solution containing neither IPNλ3 nor CCL17 and a buffer solution containing IFNλ3 and CCL17 at each predetermined concentration can be used as a calibrator for quantifying IFNλ3 and CCL17. The reagent kit of this example includes reagents each containing the capture antibody and the detection labeled antibody for IFNλ3, reagents each containing the capture antibody and the detection labeled antibody for CCL17 and a calibrator for quantifying IFNλ3 and CCL17, but in place of these reagents, reagents each containing a capture antibody for another biomarker and a detection labeled antibody for another biomarker and a calibrator may be included.
  • In a further embodiment, a container containing one reagent selected from the reagent containing a substance capable of specifically binding to IFNλ3, the reagent containing a substance capable of specifically binding to CCL17, the reagent containing a substance capable of specifically binding to CXCL11, the reagent containing a substance capable of specifically binding to IP-10, the reagent containing a substance capable of specifically binding to IL-6, and the reagent containing a substance capable of specifically binding to CXCL9 may be packed in a box and provided to a user as a reagent kit. The box may contain an attached document. Compositions, usage, storage method, etc. of the reagents may be described in the attached document. Fig. 1D shows an example of the reagent kit. In Fig.1D, 41 denotes a reagent kit, 42 denotes a container containing a reagent containing a substance capable of specifically binding to IFNλ3, 43 denotes a packing box, and 44 denotes an attached document. The reagent kit of this example includes the reagent containing a substance capable of specifically binding to IFNλ3, but in place of this reagent, reagents containing a substance capable of specifically binding to another biomarker may be included.
  • The application also provides a device for performing the prediction method and/or monitoring method of the present embodiment. Such a device is a device to assist in predicting exacerbation of respiratory infection (hereinafter, also simply referred to as "device"). Also provided herein is a computer program for making a computer execute the prediction method and/or monitoring method of the present invention. Such a computer program is a computer program to assist in predicting exacerbation of respiratory infection. Such a computer program, when executed on a computer, causes the computer to carry out the methods of the invention.
  • Hereinbelow, an example of the device for performing the prediction method of the present embodiment will be described with reference to drawings. Fig. 2 is a schematic diagram of a device of the present embodiment. A device 10 shown in Fig. 2 includes an immunoassay device 20 and a computer system 30 connected to the immunoassay device 20.
  • In the present invention, the type of immunoassay device is not particularly limited, and it can be appropriately selected according to the method for measuring a biomarker. In the example shown in Fig. 2, the immunoassay device 20 is a commercially available automated immunoassay device capable of detecting a chemiluminescent signal generated by a sandwich ELISA method using magnetic particles on which a capture antibody is immobilized and an enzyme-labeled detection antibody. The immunoassay device 20 is not particularly limited as long as it can detect a signal based on the used labeling substance, and it can be appropriately selected according to the type of the labeling substance.
  • When a reagent containing magnetic particles on which a capture antibody is immobilized, a reagent containing an enzyme-labeled detection antibody and a specimen collected from a subject are set in the immunoassay device 20, the immunoassay device 20 performs an antigen-antibody reaction using each reagent, acquires a chemiluminescent signal as optical information based on the enzyme-labeled antibody specifically bound to a biomarker, and transmits the obtained optical information to the computer system 30.
  • The computer system 30 includes a computer main body 300, an input unit 301, and a display unit 302 that displays specimen information, a determination result, and the like. The computer system 30 receives the optical information from the immunoassay device 20. Then, a processor of the computer system 30 executes a computer program to assist in predicting exacerbation of respiratory infection, installed in a hard disk 313, based on the optical information. As shown in Fig. 2, the computer system 30 may be equipment separate from the immunoassay device 20, or may be equipment including the immunoassay device 20. In the latter case, the computer system 30 may itself be the prediction assisting device 10. A commercially available automated immunoassay device may be loaded with the computer program to assist in predicting exacerbation of respiratory infection. The device 10 may be a device in which the immunoassay device 20 and the computer system 30 are integrally configured.
  • With reference to Fig. 3, the computer main body 300 includes a central processing unit (CPU) 310, a read only memory (ROM) 311, a random access memory (RAM) 312, a hard disk 313, an input/output interface 314, a reading device 315, a communication interface 316, and an image output interface 317. The CPU 310, the ROM 311, the RAM 312, the hard disk 313, the input/output interface 314, the reading device 315, the communication interface 316 and the image output interface 317 are data-communicably connected by a bus 318. The immunoassay device 20 is communicably connected to the computer system 30 via the communication interface 316.
  • The CPU 310 can execute a program stored in the ROM 311 or the hard disk 313 and a program loaded in the RAM 312. The CPU 310 calculates the measured value of the biomarker and displays it on the display unit 302.
  • The ROM 311 includes a mask ROM, PROM, EPROM, EEPROM, and the like. In the ROM 311, a computer program executed by the CPU 310 and data used for executing the computer program are recorded as described above. The computer program recorded in ROM 311 includes a basic input output system (BIOS).
  • The RAM 312 includes SRAM, DRAM, and the like. The RAM 312 is used for reading the program recorded in the ROM 311 and the hard disk 313. The RAM 312 is also used as a work area of the CPU 310 when these programs are executed.
  • In the hard disk 313, an operating system and a computer program such as an application program to be executed by the CPU 310, and data used for executing the computer program are installed.
  • The reading device 315 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid state drive, and the like. The reading device 315 can read a program or data recorded on a portable recording medium 40.
  • The input/output interface 314 includes, for example, a serial interface such as USB, IEEE1394 and RS-232C, a parallel interface such as SCSI, IDE and IEEE1284, and an analog interface including a D/A converter, an A/D converter and the like. The input unit 301 such as a keyboard and a mouse is connected to the input/output interface 314. An operator can input various commands to the computer main body 300 through the input unit 301.
  • The communication interface 316 is, for example, an Ethernet (registered trademark) interface or the like. The computer main body 300 can also transmit print data to a printer or the like through the communication interface 316.
  • The image output interface 317 is connected to the display unit 302 including an LCD, a CRT, and the like. As a result, the display unit 302 can output a video signal corresponding to the image data coming from the CPU 310. The display unit 302 displays an image (screen) according to the input video signal.
  • A processing procedure to be executed by the device 10 of the present invention will be described with reference to Fig. 4A. Here, it will be described as an example a case where a measured value of IFNλ3 is acquired from a chemiluminescent signal generated by a sandwich ELISA method using magnetic particles on which a capture antibody is immobilized, and an enzyme-labeled detection antibody and output. In place of the measured value of IFNλ3, a measured value of CCL17, CXCL11, IP-10, IL-6 or CXCL9 may be acquired.
  • In step S101, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S102, the CPU 310 calculates a measured value of IFNλ3 from the acquired optical information, and the CPU 310 stores the measured value of IFNλ3 in the hard disk 313. In step S103, the CPU 310 outputs the measured value of IFNλ3, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the measured value. When outputting the measured value of IFNλ3, a threshold value corresponding to INFλ3 may also be displayed on the display unit 302 as reference information. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • Other processing procedures to be executed by the device 10 according to an embodiment of the present invention will be described with reference to Fig. 4B. Here, a case where the measured values of IFNλ3 and CCL17 are acquired and output will be described as an example. In step S201, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S202, the CPU 310 calculates measured values of IFNλ3 and CCL17 from the acquired optical information, and the CPU 310 stores the measured values of IFNλ3 and CCL17 in the hard disk 313. In step S203, the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the measured values. When outputting the measured values of IFNλ3 and CCL17, threshold values corresponding to each of IFNλ3 and CCL17 may also be displayed on the display unit 302 as reference information. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • A flow for predicting exacerbation of respiratory infection based on the measured value of IFNλ3 will be described with reference to Fig. 4C. In step S301, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S302, the CPU 310 calculates a measured value of IFNλ3 from the acquired optical information, and the CPU 310 stores the measured value of IFNλ3 in the hard disk 313. In step S303, the CPU 310 compares the calculated measured value of IPNλ3 with a threshold value corresponding to IFNλ3 stored in the hard disk 313. When the measured value of IFNλ3 is greater than or equal to the threshold value, the process proceeds to step S304. In step S304, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • On the other hand, in step S303, when the measured value of IFNλ3 is less than the threshold value, the process proceeds to step S305. In step S305, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. In step S306, the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • A flow for predicting exacerbation of respiratory infection based on the measured values of IFNλ3 and CXCL11 will be described with reference to Fig. 4D. In Fig. 4D, the threshold value corresponding to the measured value of IFNλ3 is referred to as "first threshold value", and the threshold value corresponding to the measured value of CXCL11 is referred to as "second threshold value".
  • In step S401, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S402, the CPU 310 calculates measured values of IFNλ3 and CXCL11 from the acquired optical information, and the CPU 310 stores the measured values of IFNλ3 and CXCL11 in the hard disk 313. In step S403, the CPU 310 compares the calculated measured value of IFNλ3 with the first threshold value stored in the hard disk 313. When the measured value of IFNλ3 is greater than or equal to the first threshold value, the process proceeds to step S404. In step S404, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • In step S403, when the measured value of IFNλ3 is less than the first threshold value, the process proceeds to step S405. In step S405, the CPU 310 compares the calculated measured value of CXCL11 with the second threshold value stored in the hard disk 313. When the measured value of CXCL11 is greater than or equal to the second threshold value, the process proceeds to step S404, and the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. In step S405, when the measured value of CXCL11 is less than the second threshold value, the process proceeds to step S406. In step S406, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. In step S407, the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • A flow for predicting exacerbation of respiratory infection based on the measured values of IFNλ3 and CCL17 will be described with reference to Fig. 4E. In Fig. 4E, the threshold value corresponding to the measured value of IFNλ3 is referred to as "first threshold value", and the threshold value corresponding to the measured value of CCL17 is referred to as "third threshold value".
  • In step S501, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S502, the CPU 310 calculates measured values of IFNλ3 and CCL17 from the acquired optical information, and the CPU 310 stores the measured values of IFNλ3 and CCL17 in the hard disk 313. In step S503, the CPU 310 compares the calculated measured value of IFNλ3 with the first threshold value stored in the hard disk 313. When the measured value of IFNλ3 is greater than or equal to the first threshold value, the process proceeds to step S504. In step S504, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. In step S503, when the measured value of IFNλ3 is less than the first threshold value, the process proceeds to step S505. In step S505, the CPU 310 compares the calculated measured value of CCL17 with the third threshold value stored in the hard disk 313. When the measured value of CCL17 is greater than or equal to the third threshold value, the process proceeds to step S506. In step S506, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low.
  • In step S505, when the measured value of CCL17 is less than the third threshold value, the process proceeds to step S504, and the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. In step S507, the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • A flow for predicting exacerbation of respiratory infection based on the measured value of CCL17 will be described with reference to Fig. 4F. In step S601, the CPU 310 acquires optical information (chemiluminescent signal) from the immunoassay device 20. In step S602, the CPU 310 calculates a measured value of CCL17 from the acquired optical information, and the CPU 310 stores the measured value of CCL17 in the hard disk 313. In step S603, the CPU 310 compares the calculated measured value of CCL17 with the threshold value corresponding to CCL17 stored in the hard disk 313. When the measured value of CCL17 is less than the threshold value, the process proceeds to step S604. In step S604, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high.
  • On the other hand, in step S603, when the measured value of CCL17 is greater than or equal to the threshold value, the process proceeds to step S605. In step S605, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. In step S606, the CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result. As a result, it is possible to provide a doctor or the like with an index to assist in predicting exacerbation of respiratory infection.
  • In another embodiment, three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these three biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured values of all biomarkers are less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • In another embodiment, three selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these three biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured value of at least biomarker is less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • In another embodiment, four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these four biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured values of all biomarkers are less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • In another embodiment, four selected from the group consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for these four biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured value of at least biomarker is less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • In another embodiment, five consisting of IFNλ3,CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for five biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured value of at least one biomarker is greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured values of all biomarkers are less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • In another embodiment, five consisting of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 are used as biomarkers. In this case, the CPU 310 acquires optical information (chemiluminescence signal) from the immunoassay device 20 for five biomarkers. The CPU 310 calculates a measured value of each biomarker from the acquired optical information. The CPU 310 stores the measured value of each biomarker in the hard disk 313. The CPU310 compares the measured value of each biomarker with the corresponding threshold value, and when the measured values of all biomarkers are greater than or equal to the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is high. When the measured value of at least biomarker is less than the threshold value, the CPU 310 stores in the hard disk 313 a determination result that the possibility of exacerbation of respiratory infection of the subject is low. The CPU 310 outputs the determination result, and the CPU 310 displays the determination result on the display unit 302 or makes a printer to print the determination result.
  • One embodiment relates to a method for treating a respiratory infection. The method for treating a respiratory infection of the present invention includes measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, predicting exacerbation of respiratory infection based on a measured value of the biomarker, and performing medical intervention on a subject predicted to exacerbate respiratory infection in the predicting. Examples of the "medical intervention" include drug administration, surgery, immunotherapy, gene therapy, oxygenation procedures, heart-lung machine procedures, and the like. The drug can be appropriately selected from known therapeutic drugs for respiratory infections or candidate medicines therefor. For example, when the respiratory infection is SARS-CoV-2 infection, examples of the drug include drugs having an antiviral action, drugs that reduce inflammation, ACE inhibitors, and the like. Specific examples of the drug include favipiravir, lopinavir, ritonavir, nafamostat, camostat, remdesivir, ribavirin, ivermectin, ciclesonide, chloroquine, hydroxychloroquine, interferon, tocilizumab, sarilumab, tofasitinib, baricitinib, ruxolitinib, acalabrutinib, ravulizumab, eritoran, ibudilast, HLCM051, LY3127804, and the like.
  • Hereinafter, the present disclosure will be described in more detail by way of examples. Hereinafter, "HISCL" refers to a registered trademark of Sysmex Corporation.
    • EXAMPLES Example 1 (1) Biological sample
  • Serum obtained from 8 patients whose SARS-CoV-2 infection was confirmed by PCR test was used as a biological sample. The serum was prepared from blood collected at a plurality of time point from the day the patient was hospitalized. Information on each patient is shown in Table 1. In the table, "onset date" indicates the day when cold symptoms such as fever and cough appeared. "Severity" indicates a final medical condition of each patient after hospitalization. "Mild" indicates a case without pneumonia. "Moderate" indicates a case with pneumonia without oxygen demand. "Severe" indicates a case with pneumonia with oxygen demand. "Critical" indicates a case requiring intensive care management including mechanical ventilation management. [Table 1]
    Case No. Onset date Hospitalization date Age Sex Severity
    4 2020/1/31 2020/1/31 41 M moderate
    5 2020/1/30 2020/1/30 50 F severe
    7 2020/2/5 2020/2/11 63 M critical
    8 2020/2/8 2020/2/11 28 F mild
    9 2020/1/27 2020/2/12 83 M severe
    10 2020/2/7 2020/2/12 64 F mild
    11 2020/2/6 2020/2/13 63 M critical
    12 2020/2/13 2020/2/17 53 M severe
    • (2) Measurement of biomarkers (2.1) Measurement of chemokines and cytokines
  • Concentrations of various chemokines and cytokines were measured using Bio-Plex Pro (trademark) Human Chemokine 40-Plex Panel (#171AK99MR2, Bio-Rad Laboratories, Inc.) and Bio-Plex Pro (trademark) Human Cytokine Screening 48-Plex Panel (#12007283, Bio-Rad Laboratories, Inc.). A Bio-Plex MAGPIX system (Bio-Rad Laboratories, Inc.) was used as a measuring device. Specific operation was performed according to an attached document of the kits and an attached document of the measuring device.
    • (2.2) Measurement of INFλ3 (IL-28B)
  • Concentration of INFλ3 as a cytokine not included in measurement items of the above kits was measured by a fully automatic immunoassay device HISCL-5000 (Sysmex Corporation) using the following R1 to R5 reagents. An R1 reagent (capture antibody reagent) was prepared by labeling an anti-hIL-28B antibody (clone name: Hyb-TA2650B) provided by the National Center for Global Health and Medicine with biotin by a conventional method, and dissolving the labeled antibody in 1% bovine serum albumin (BSA) and a 0.5% casein-containing buffer. As an R2 reagent (solid phase), a HISCL (registered trademark) R2 reagent (Sysmex Corporation) containing streptavidin-coupled magnetic particles was used. An R3 reagent (detection antibody reagent) was prepared by making an anti-rhIL-28B antibody (clone name: Hyb-TA2664) provided by the National Center for Global Health and Medicine into a Fab' fragment by a conventional method, labeling this Fab' fragment with ALP by a conventional method, and dissolving the labeled antibody in 1% BSA and a 0.5% casein-containing buffer. As an R4 reagent (measurement buffer solution), a HISCL R4 reagent (Sysmex Corporation) was used. As an R5 reagent (ALP substrate solution), a HISCL R5 reagent (Sysmex Corporation) was used. A method for preparing an anti-hIL-28B antibody and an anti-rhIL-28B antibody is described in Japanese Examined Patent No. 6081699 .
  • A measurement procedure according to HISCL-5000 was as follows. After mixing serum (30 µL) and the R1 reagent (100 µL), the R2 reagent (30 µL) was added thereto. The magnetic particles in the obtained mixed solution were magnetically collected to remove the supernatant, and a HISCL washing solution (300 µL) was added to wash the magnetic particles. The supernatant was removed, and the R3 reagent (100 µL) was added to the magnetic particles and mixed. The magnetic particles in the obtained mixed solution were magnetically collected to remove the supernatant, and a HISCL washing solution (300 µL) was added to wash the magnetic particles. Supernatant was removed, and the R4 reagent (50 µL) and the R5 reagent (100 µL) were added to the magnetic particles, and the chemiluminescence intensity was measured. As a calibrator (antigen for preparing a calibration curve), hIL-28B (10-046) provided by the National Center for Global Health and Medicine was used. The calibrator was measured in the same manner as serum to prepare a calibration curve. The chemiluminescence intensity obtained by the measurement of each serum was applied to the calibration curve to determine the concentration of INFλ3.
    • (3) Measurement results
  • From the measurement results of various chemokines and cytokines, IFNλ3, CXCL11, IP-10, IL-6, CXCL9 and CCL17 were found as biomarkers that can predict prognosis of SARS-CoV-2 infection. Figs. 5 to 10 show graphs plotting measured values of each patient for IFNλ3,CXCL11, IP-10, IL-6, CXCL9 and CCL17. In the figures, "elapsed days" indicates the number of days from the day when the patient was hospitalized (elapsed days 0). Arrows in the figures indicate a time point when the patient was treated with an oxygen inhaling apparatus or a heart-lung machine. In the figures, "IFNL3" means IFNλ3.
  • As can be seen from A to E in Figs. 5 to 9, patients in case Nos. 5, 7, 9, 11 and 12 tended to exacerbate infection after IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 showed high values. The patient in case No. 9 had relatively low measured values of IPNλ3 as compared to other severely ill patients, but tended to have high measured values of CXCL11, IP-10, IL-6 and CXCL9. On the other hand, as can be seen from F, G and H in Figs. 5 to 9, patients in case Nos. 4, 8 and 10 tended to show low measured values of IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 as compared to severely ill patients. These results suggested that IFNλ3, CXCL11, IP-10, IL-6 and CXCL9 can be used as biomarkers to predict exacerbation of the respiratory infection.
  • As can be seen from Figs. 10A to 10H, the measured values of CCL17 tended to show high values in mildly ill patients as compared to severely ill patients. This result suggested that CCL17 can be used as a biomarker to find a patient with the possibility of exacerbation of respiratory infection of the subject is low, by using in combination with IFNλ3, CXCL11, IP-10, IL-6 or CXCL9.
    • Example 2 (1) Biological sample
  • Serum obtained from 20 patients whose SARS-CoV-2 infection was confirmed by PCR test was used as a biological sample. The serum was prepared from blood collected at a plurality of time point from the day the patient was hospitalized. Severity of the 20 patients was mild in 2, moderate in 11, severe in 2, and critical in 5.
    • (2) Measurement of biomarkers
  • Concentrations of IFNλ3 and CCL17 in the plasma of each patient were measured in the same manner as in Example 1. Figs. 11 and 12 show graphs plotting the measured values of 28 patients, including 20 patients in Example 2 and 8 patients in Example 1, for IFNλ3 and CCL17. Fig. 11A shows IFNλ3 measured values for critical and severe patients, and Fig. 11B shows IPNλ3 measured values for moderate and mild patients. Fig. 12A shows CCL17 measured values for critical and severe patients, and Fig. 12B shows CCL17 measured values for moderate and mild patients. In the figures, "Days after hospitalization" indicates the number of days from the day when the patient was hospitalized (0 day).
    • (3) Analysis of measured values of biomarkers
  • Analysis was performed on the measured values of 28 patients, including 20 patients in Example 2 and 8 patients in Example 1. Of the 28 patients, patients with mild or moderate severity were classified as "low risk group" (hereinafter referred to as "L group"), patients with severe or critical severity were classified as "high risk group" (hereinafter referred to as "H group"), and concentrations of IFNλ3 and CCL17 in each group were plotted. The results are shown in Figs. 13A and 13B. Horizontal lines in the figures indicate first quartile, median and third quartile of the biomarker concentration of each group.
  • The biomarker concentrations of 28 patients were analyzed by ROC, and an optimum cutoff value (threshold value) for distinguishing between the L group and the H group was set. For 28 patients, the set cutoff values were used to calculate sensitivity, specificity and area under the curve (AUC) of determination when determining whether or not the possibility of exacerbation of respiratory infection is high. The obtained ROC curves are shown in Figs. 14A and 14B. The cutoff values of IFNλ3 and CCL17, and the sensitivity, specificity, AUC and p-values of determination using the cutoff values are shown in Table 2. Event-free survival (EFS) after hospitalization was examined by Kaplan-Meier method for two patient groups classified based on the cutoff values. The obtained Kaplan-Meier curves are shown in Figs. 15A and 15B. [Table 2]
    Biomarker Cutoff value (pg/mL) Sensitivity (%) Specificity (%) AUC p
    INF λ3 13.55 91.67 (64.61-99.57) 93.75 (71.67-99.68) 0.96 (0.89-1.0) <0.001
    CCL17 87.45 100 (75.75-100) 100 (80.64-100) 1.0 (1.0-1.0) <0.001
  • It was shown from Table 2 that IFNλ3 and CCL17 are biomarkers that allow prediction of exacerbation of respiratory infection. It was shown from Fig. 15A that the possibility of exacerbation of respiratory infection is high when the measured value of IFNλ3 is greater than the cutoff value, and the possibility of exacerbation of respiratory infection is low when the measured value of IFNλ3 is less than or equal to the cutoff value. It was shown from Fig. 15B that the possibility of exacerbation of respiratory infection is high when the measured value of CCL17 is less than the cutoff value, and the possibility of exacerbation of respiratory infection is low when the measured value of CCL17 is greater than or equal to the cutoff value.
    Figure imgb0001
    Figure imgb0002
    Figure imgb0003
    Figure imgb0004
    Figure imgb0005

Claims (16)

  1. A method for assisting prediction of exacerbation of respiratory infection, comprising: measuring a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and a measured value of the biomarker is an index to predict exacerbation of the respiratory infection.
  2. The method according to claim 1, wherein the biomarker is IFNλ3, CXCL11, IP-10, IL-6 or CXCL9, and
    when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  3. The method according to claim 2, wherein
    when the measured value of the biomarker is less than the threshold value corresponding to the biomarker, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  4. The method according to claim 1, wherein the biomarker is CCL17, and
    when the measured value of CCL17 is less than a threshold value corresponding to CCL17, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  5. The method according to claim 4, wherein
    when the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  6. The method according to any one of claims 1 to 5, wherein the index is a temporal change in the measured value of the biomarker in the subject.
  7. The method according to any one of claims 1 to 6, wherein the specimen is whole blood, plasma or serum.
  8. The method according to any one of claims 1 to 7, wherein the respiratory infection is an infection caused by a virus, particularly .
  9. The method according to claim 8, wherein the virus is α-coronavirus, β-coronavirus, γ-coronavirus or δ-coronavirus.
  10. The method according to claim 9, wherein
    the β-coronavirus is any one of SARS-CoV-2, HCoV-OC43, HCoV-HKU1, SARS-CoV, Bat SL-CoV-WIV1, BtCoV-HKU4, BtCoV-HKU5, MERS-CoV and BtCoV-HKU9.
  11. A method for monitoring a measured value of a biomarker in a specimen collected from a subject suffering from a respiratory infection or a subject suspected of having a respiratory infection, comprising: acquiring measured values of the biomarker using specimens collected from the subject at a plurality of time points, wherein the biomarker is at least one selected from the group consisting of IFNλ3, CCL17, CXCL11, IP-10, IL-6 and CXCL9, and the measured values are indices to predict exacerbation of the respiratory infection.
  12. The method according to claim 11, wherein the biomarker is IFNλ3, CXCL11, IP-10, IL-6 or CXCL9, and
    when the measured value of the biomarker is greater than or equal to a threshold value corresponding to the biomarker at at least one of the plurality of time points, a possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  13. The method according to claim 12, wherein
    when the measured value of the biomarker is less than the threshold value corresponding to the biomarker at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  14. The method according to claim 11, wherein the biomarker is CCL17, and
    when the measured value of CCL17 is less than a threshold value corresponding to CCL17 at at least one of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be high.
  15. The method according to claim 14, wherein
    when the measured value of CCL17 is greater than or equal to the threshold value corresponding to CCL17 at any of the plurality of time points, the possibility of exacerbation of respiratory infection of the subject is suggested to be low.
  16. Use of a reagent kit in the method according to any one of claims 1 to 15, the reagent kit comprising a reagent comprising a substance capable of specifically binding to the biomarker.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01254868A (en) 1988-04-05 1989-10-11 Eiji Ishikawa Method of measuring antigen material with extra-high sensitivity
JP6081699B2 (en) 2011-12-28 2017-02-15 雅史 溝上 Method for analyzing IL-28B

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01254868A (en) 1988-04-05 1989-10-11 Eiji Ishikawa Method of measuring antigen material with extra-high sensitivity
JP6081699B2 (en) 2011-12-28 2017-02-15 雅史 溝上 Method for analyzing IL-28B

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KAO-JEAN HUANG ET AL: "An interferon-?-related cytokine storm in SARS patients", JOURNAL OF MEDICAL VIROLOGY, vol. 75, no. 2, 1 January 2004 (2004-01-01), pages 185 - 194, XP055183753, ISSN: 0146-6615, DOI: 10.1002/jmv.20255 *
KIM EU SUK ET AL: "Clinical Progression and Cytokine Profiles of Middle East Respiratory Syndrome Coronavirus Infection", JOURNAL OF KOREAN MEDICAL MEDICINE, vol. 31, no. 11, 1 January 2016 (2016-01-01), SEOUL, KR, pages 1717 - 1725, XP055812552, ISSN: 1011-8934, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056202/pdf/jkms-31-1717.pdf> DOI: 10.3346/jkms.2016.31.11.1717 *
LIU YINGXIA ET AL: "Elevated plasma levels of selective cytokines in COVID-19 patients reflect viral load and lung injury", NATIONAL SCIENCE REVIEW, vol. 7, no. 6, 9 March 2020 (2020-03-09), pages 1003 - 1011, XP055825706, ISSN: 2095-5138, Retrieved from the Internet <URL:http://academic.oup.com/nsr/article-pdf/7/6/1003/38881910/nwaa037.pdf> DOI: 10.1093/nsr/nwaa037 *
SUGIYAMA MASAYA ET AL: "Serum CCL17 level becomes a predictive marker to distinguish between mild/moderate and severe/critical disease in patients with COVID-19", GENE, vol. 766, 1 January 2021 (2021-01-01), NL, pages 145145, XP055836615, ISSN: 0378-1119, DOI: 10.1016/j.gene.2020.145145 *

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